Methods of purifying recombinant ADAMTS13 and other proteins and compositions thereof

ABSTRACT

Provided herein are methods for purifying recombinant A Disintegrin-like and Metallopeptidase with Thrombospondin Type 1 Motif 13 (ADAMTS13) protein from a sample. The method comprises enriching for ADAMTS13 protein by chromatographically contacting the sample with hydroxyapatite under conditions that allow ADAMTS13 protein to appear in the eluate or supernatant from the hydroxylapatite. The methods may further comprise tandem chromatography with a mixed mode cation exchange/hydrophobic interaction resin that binds ADAMTS13 protein. Additional optional steps involve ultrafiltration/diafiltration, anion exchange chromatography, cation exchange chromatography, and viral inactivation. Also provided herein are methods for inactivating virus contaminants in protein samples, where the protein is immobilized on a support. Also provided herein are compositions of ADAMTS13 prepared according to said methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/230,308, filed Jul. 31, 2009, which is hereby incorporated byreference in its entirety, and to which application we claim priority.

FIELD OF INVENTION

The present invention relates generally to methods of purifyingrecombinant A Disintegrin-Like And Metallopeptidase with ThromboSpondinType 1 Motif 13 (ADAMTS13) and other proteins, and compositionscomprising such purified proteins.

BACKGROUND OF THE INVENTION

The metalloproteinase gene family, ADAM (a disintegrin andmetalloproteinase), includes members that are membrane-anchoredproteases with diverse functions. ADAMTS family members aredistinguished from ADAMs by the presence of one or more thrombospondin1-like (TSP1) domain(s) at the C-terminus and the absence of the EGFrepeat, transmembrane domain and cytoplasmic tail typically observed inADAM metalloproteinases.

A Disintegrin-Like and Metallopeptidase with Thrombospondin Type 1 Motif13 (ADAMTS13) is a member of the ADAMTS family. ADAMTS13 has eightthrombospondin domains and no hydrophobic transmembrane domain.Accordingly, it is secreted. ADAMTS13 cleaves von Willebrand Factor atthe Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ bond and requires both calcium and zinc ions tofunction. ADAMTS13 is also known as “von Willebrand Factor-CleavingProtease” and “VWFCP.”

Deficient ADAMTS13 expression has been implicated in the pathogenesis ofsome diseases, e.g., thrombotic disorders such as thromboticthrombocytopenic purpura (TTP) (see, e.g., U.S. Patent Publication No.20070015703). In TTP, deficiency and/or inhibition of ADAMTS13 resultsin extensive microscopic thomboses that form in small blood vesselsthroughout the body (thrombotic microangiopathy). Red blood cellspassing through the microscopic clots experience shear stress, whichcauses damage to the red blood cell membrane, and which in turn leads tointravascular hemolysis and schistocyte formation. Thromboses also causereduced blood flow, which can result in end organ damage. Symptomstypically include neurological problems, such as hallucination, bizarrebehavior, altered mental status, stroke, or headaches; kidney failure;fever; and thrombocytopenia (low platelet count), resulting in bruisingor purpura; and microangiopathic hemolytic anemia, involving anemia andjaundice. Current therapy involves plasmapheresis to reduce circulatingantibodies against ADAMTS13, and/or replenishing blood levels of theenzyme.

Therefore, a strong need exists for providing methods of purifyingrecombinant ADAMTS13, particularly on a commercial production scale,which may be used as a therapeutic agent. Purification of ADAMTS13 hasproven difficult and various approaches have been attempted, includingchromatography. A chromatographic material that binds non-ADAMTS13protein, allowing the ADAMTS13 protein to appear in the eluate orsupernatant, would provide a useful approach for purification. Achromatography material that binds ADAMTS13 protein, while non-ADAMTS13impurities either remain in solution or bind much more strongly, alsopresents an attractive approach, and may be used in tandem with otherapproaches. The instant disclosure provides such approaches.

Furthermore, virus contaminants have posed additional challenges in thepurification of ADAMTS13 proteins, as well as other proteins andrecombinant proteins. One conventional approach involved treating asample to be purified with a solvent-detergent mixture in solution.Incubation of the sample with the solvent-detergent chemicals led todeactivation of lipid-coated viruses. This in-solution treatment,however, inefficiently required transfer of the sample to at least oneother vessel, e.g., to facilitate removal of the solvent-detergentchemicals after treatment. Further, some proteins including ADAMTS13 aresensitive to the solvent-detergent chemicals, resulting in aggregateformation. The instant disclosure provides an approach involvingimmobilization of the protein during the solvent-detergent treatment toaddress such problems of virus inactivation.

SUMMARY OF INVENTION

One aspect of the invention relates to a method for purifyingrecombinant A Disintegrin-like and Metallopeptidase with ThrombospondinType 1 Motif 13 (ADAMTS13) protein (particularly, human ADAMTS13) from asample comprising ADAMTS13 protein and non-ADAMTS13 impurities. It hassurprisingly been found that hydroxyapatite chromatography can be usedunder conditions suitable for purifying ADAMTS13 protein fromnon-ADAMTS13 impurities. The method comprises enriching for ADAMTS13protein by chromatographically contacting the sample with hydroxyapatiteunder conditions that allow ADAMTS13 protein to appear in an eluate fromthe hydroxyapatite. That is, the sample is subjected to chromatographywith hydroxyapatite under conditions that allow the ADAMTS13 protein,preferably a substantial portion of the ADAMTS13 protein, to not bindthe hydroxyapatite, while impurities are retained. In some preferredembodiments, recombinant ADAMTS13 protein is purified from supernatantcollected from culturing CHO cells comprising recombinant ADAMTS13nucleic acid. In some preferred embodiments, the percent yield in thesupernatant or eluate surprisingly is 50% to 100%. The method mayfurther comprise tandem chromatography comprising chromatographicallycontacting the eluate from the hydroxyapatite with a mixed mode cationexchange/hydrophobic interaction resin that binds ADAMTS13 protein. Insome preferred embodiments, the percent yield ADAMTS13 after enrichmentby tandem chromatography is surprisingly at least 60%.

In some embodiments, the method further comprises an optionalpre-enrichment preparation step to concentrate ADAMTS13 in the sampleand/or bind the ADAMTS13 protein to anion exchange resin. For example,the method may further comprise chromatographically contacting thesample with an anion exchange resin and eluting the ADAMTS13 proteinfrom the anion exchange resin before chromatographic contact with thehydroxyapatite; and/or concentrating the ADAMTS13 protein in the sampleby ultrafiltration before chromatographic contact with thehydroxyapatite; and/or stabilizing the ADAMTS13 protein by diafiltrationexchange into a buffer comprising calcium ions and zinc ions beforechromatographic contact with the hydroxyapatite. In some preferredembodiments, the sample is concentrated by 10-fold to 20-foldultrafiltration, the buffer exchanged by diafiltration with a molecularcut-off of 30 kDa to a low-conductivity buffer containing calcium andzinc ions, and the ADAMTS13 bound and eluted from an anion exchangeresin, such as ANX Sepharose Fast Flow, POROS 50D, or POROS 50PI, priorto tandem chromatography. The eluate pool from the anion exchangechromatography step is in some preferred embodiments diluted 1:4 withhydroxyapatite-dilution buffer to reduce conductivity to 6 mS/cm beforetandem chromatography with hydroxyapatite, comprising chromatographywith hydroxyapatite followed by chromatography using the eluate from thehydroxyapatite with a mixed mode cation exchange/hydrophobic interactionresin that binds ADAMTS13 protein. In some preferred embodiments, theeluate from the pre-enrichment step(s) can surprisingly provide apercent yield of at least 75%.

In some embodiments, the method further comprises an optional polishingstep by cation exchange chromatography, following chromatographiccontact with the hydroxyapatite or the mixed mode resin. In suchembodiments, following contact with the hydroxyapatite or the cationexchange/hydrophobic interaction resin, the method may further comprisea step of preparing the ADAMTS13 protein for cation exchange by reducingbuffer conductivity. In some embodiments, this preparing step isperformed by ultrafiltration/diafiltration, by dialysis, and/or by gelfiltration. In some embodiments where ultrafiltration/diafiltration isused, the cut-off is 10 kDa. In some embodiments, the buffer exchange iscarried out by anion exchange chromatography on ANX Sepharose-FF lowsub. In some embodiments where dialysis is used, the dialysis mayconsist of no more than 2 passes through a single dialysis module. Insome preferred embodiments, the cation exchange chromatography iscarried out on a Source S column or POROS S column. In some preferredembodiments, the percent yield ADAMTS13 after reducing the bufferconductivity is surprisingly at least 90%, and after polishing by cationexchange chromatography, surprisingly at least 70%.

In some embodiments, the method further comprises subjecting theADAMTS13 protein to an optional virus inactivation step, e.g., todeactivate viruses and/or remove viruses and viral particles. In someembodiments, the virus inactivation step comprises adding asolvent-detergent mixture comprising a non-ionic detergent and anorganic solvent to the ADAMTS13 protein. In some preferred embodiments,the ADAMTS13 protein is immobilized, e.g., immobilized on a cationexchange resin. In some embodiments, the solvent-detergent mixturecomprises 1% TRITONX-100, 0.3% Tri-N-butyl phosphate, and 0.3% TWEEN 80;and/or solvent-detergent treatment lasts for 30 minutes at 12° C. to 16°C. Alternatively or in addition, the virus inactivation step maycomprise filtering the ADAMTS13 protein with a nanofilter to removeviruses and/or viral particles. In some such embodiments, nanofiltrationis carried out through a 20 N or 35 N filter, before and/or aftersolvent-detergent treatment. In some embodiments, the virus inactivationstep is performed after the preparing step, described above, and/orafter the tandem chromatography step; and/or after polishing cationexchange chromatography, described above. In some preferred embodiments,the percent yield ADAMTS13 after virus inactivation is surprisingly atleast 95%.

In some embodiments, the method further comprises eluting the ADAMTS13protein from the cation exchange resin. In some preferred embodiments,gradient elution is used, e.g., gradient elution comprising a firstbuffer having low salt concentration and a second buffer having highersalt concentration. In some more preferred embodiments, step elution isused, even more preferably, the step elution involves elution theADAMTS13 protein from the resin with a storage buffer. For example, thestorage buffer may have a pH of greater than 7.0 and comprise less than10 mM calcium ions, a buffering compound, 0.05% non-ionic detergent, anda salt. In some more preferred embodiments, the method comprises nosubsequent concentration or buffer exchange step, following elution fromthe resin with the storage buffer.

In a particularly preferred embodiment, a method for purifyingrecombinant ADAMTS13 protein from a sample comprising ADAMTS13 proteinand non-ADAMTS13 impurities is provided, the method comprisingchromatographically contacting the sample with hydroxyapatite underconditions that allow the ADAMTS13 protein to appear in an eluate or asupernatant from the hydroxyapatite; and then chromatographicallycontacting said eluate with a cation exchange/hydrophobic interactionresin that binds the ADAMTS13 protein, preferably as tandemchromatography.

In another particularly preferred embodiment, chromatography stepsdescribed above are preceded by chromatographically contacting thesample with an anion exchange resin and eluting the ADAMTS13 proteinfrom the anion exchange resin; and/or concentrating the ADAMTS13 proteinin the sample by ultrafiltration, and stabilizing the ADAMTS13 proteinby diafiltration exchange into a buffer comprising calcium ions and zincions before chromatographic contact with the hydroxyapatite.

In another particularly preferred embodiment, following contact with thehydroxyapatite or the cation exchange/hydrophobic interaction resin, themethod further comprises the step of preparing the ADAMTS13 protein forcation exchange by reducing buffer conductivity, wherein the preparingstep is performed by ultrafiltration/diafiltration; and/or by dialysisconsisting of no more than 2 passes through a single dialysis module;and/or by gel filtration.

In still another particularly preferred embodiment, the method comprisesobtaining a sample from supernatant collected from culturing CHO cellscomprising recombinant ADAMTS13 nucleic acid; chromatographicallycontacting the sample with an anion exchange resin and eluting theADAMTS13 protein from the anion exchange resin before chromatographiccontact with the hydroxyapatite; and/or concentrating the ADAMTS13protein in the sample by ultrafiltration; and stabilizing the ADAMTS13protein by diafiltration exchange into a buffer comprising calcium ionsand zinc ions before chromatographic contact with the hydroxyapatite;followed by chromatographically contacting the sample withhydroxyapatite under conditions that allow the ADAMTS13 protein toappear in an eluate or a supernatant from the hydroxyapatite; and thenchromatographically contacting the eluate with a cationexchange/hydrophobic interaction resin that binds the ADAMTS13 protein;followed by preparing the ADAMTS13 protein for cation exchange byreducing buffer conductivity, e.g., by ultrafiltration/diafiltration;and/or by dialysis consisting of no more than 2 passes through a singledialysis module; and/or by gel filtration, optionally further comprisingone or more virus inactivation steps. In some such embodiments, thevirus inactivation step comprises adding a solvent-detergent mixturecomprising a non-ionic detergent and an organic solvent to the ADAMTS13protein, wherein the ADAMTS13 protein is immobilized on a cationexchange resin and the solvent-detergent mixture comprises 1%TRITONX-100, 0.3% Tri-N-butyl phosphate, and 0.3% TWEEN 80. In stillanother embodiment, the virus inactivation step uses, as well as orinstead of the solvent-detergent treatment, a nanofilter to removeviruses and/or viral particles. In some preferred embodiments, thepercent yield ADAMTS13 of the overall procedure outlined above issurprisingly 22-24% or more, and in even more preferred embodiments,aggregates surprisingly are reduced by 50%.

In some embodiments where the ADAMTS13 has been immobilized on a cationexchange resin, the method further comprises eluting the ADAMTS13protein from the resin using step elution with a storage buffer having apH of greater than 7.0 and comprising less than 10 mM calcium ions, abuffering compound, 0.05% non-ionic detergent, and a salt; or usinggradient elution comprising a first buffer having low salt content and asecond buffer having higher salt content.

Another aspect of the invention relates to a composition comprising arecombinant ADAMTS13 protein prepared according to any embodiment of themethods described herein. In some embodiments, the composition is apharmaceutical composition, e.g., a composition comprising purifiedADAMTS13 protein and a pharmaceutically acceptable carrier.

Still a further aspect of the invention relates to a method forinactivating virus contaminants in a protein sample, where the proteinmay be any protein from a source that may have viral contaminants. Inpreferred embodiments, the protein is recombinant protein, particularlyproteins sensitive to aggregation when exposed to organic solvents anddetergents. In some embodiments, the protein may be ADAMTS13 protein, inparticular recombinant ADAMTS13, or a different protein (in particular,a different recombinant protein). In some embodiments, the recombinantprotein is a blood coagulation factor. In some embodiments, the proteinis, e.g., one or more of Factor VIII, Factor II, Factor VIIa, Factor IX,thrombin, von Willebrand factor, anti-MIF antibody, or another proteinbeing purified by chromatography. The viral inactivation may be carriedout in conjunction with protein purification or not. In someembodiments, the method comprises immobilizing the protein on a support;and treating the immobilized protein with a detergent-solvent mixturecomprising a non-ionic detergent and an organic solvent. In somepreferred embodiments, the support is a chromatographic resin. In evenmore preferred embodiments, the detergent-solvent mixture comprises 1%Triton X-100, 0.3% Tri-N-butyl phosphate, and 0.3% Polysorbate 80 (Tween80). The solvent-detergent mixture treatment can continue for aprolonged time, e.g., for 30 minutes to 1 hour, while the proteinremains immobilized on the chromatographic resin, e.g., on a cationexchange resin; and/or solvent-detergent treatment may occur at 2° C. to10° C. This approach to virus inactivation surprisingly can reduce theformation of protein aggregates during treatment with adetergent-solvent mixture by a significant amount, e.g., by more than50%, as compared to treatment with a solvent-detergent mixture while theprotein is not immobilized in solution. In some preferred embodiments,the procedure is followed by eluting the protein from the support with abuffer, such as gradient elution where small amounts of aggregates thatdo form are further removed in the late eluting fraction. In somepreferred embodiments, the procedure is followed by eluting the proteinwith a storage buffer. In some more preferred embodiments, the elutionbuffer comprises a concentration of 0.1% Tween 80. In some even morepreferred embodiments, the method comprises no subsequent concentrationor buffer exchange step, following elution from the resin with thestorage buffer. In some preferred embodiments, aggregates surprisinglyare reduced by 50%.

In yet still another particularly preferred embodiment, a method forinactivating virus contaminants in a protein sample is provided, themethod comprising immobilizing the protein on a chromatographic resin;and treating the immobilized protein with a solvent-detergent mixturecomprising 1% Triton X-100, 0.3% Tri-N-butyl phosphate, and 0.3%Polysorbate 80, for 30 minutes to 1 hour. In some such embodiments, themethod further comprises eluting the protein with a storage bufferhaving a pH of greater than 7.0 and comprising less than 10 mM calciumions, a buffering compound, 0.05% non-ionic detergent, and a salt.

These and other aspects of the invention are described in more detailbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flow diagram of exemplary steps of the method forpurifying recombinant a disintegrin-like and metallopeptidase withthrombospondin type 1 motif 13 (ADAMTS13) from a sample comprisingADAMTS13 and non-ADAMTS13 impurities, in accordance with the instantinvention. The order of the steps set forth in FIG. 1 may be re-ordered,and/or one or more steps omitted, as disclosed herein and as understoodby one of skill in the art.

FIGS. 2A-D depict variations in purification runs on a cation exchangecolumn. FIG. 2A depicts a procedure involving cation exchangechromatography with step elution, following virus inactivation; FIG. 2Bdepicts a procedure involving cation exchange chromatography with stepelution, but without a preceding virus inactivation; FIG. 2C depicts aprocedure involving cation exchange chromatography with gradientelution, followed by virus inactivation on the chromatographic column;and FIG. 2D depicts a procedure involving cation exchange chromatographywith gradient elution, but without a preceding virus inactivation.

DETAILED DESCRIPTION

One aspect of the invention relates to a method for the purification ofrecombinant A Disintegrin-Like and Metallopeptidase with ThrombospondinType 1 Motif 13 (ADAMTS13) protein from a sample, which may alsocomprise non-ADAMTS13 impurities. The protein sample may also includevirus contaminants, which may be removed and/or inactivated by one ormore virus inactivation steps.

As used herein “A Disintegrin-Like and Metallopeptidase withThrombospondin Type 1 Motif 13,” “ADAMTS13,” “ADAMTS13 protein,”“ADAMTS13 polypeptide,” and “recombinant ADAMTS13” are interchangeable(unless otherwise specified) and refer to a recombinant mammalianADAMTS13 protein, which also may be a biologically active derivative orfragment of a full-length ADAMTS13 protein. The amino acid sequence offull-length human and murine ADAMTS13 proteins have respectiveUniProtKB® accession numbers of Q76LX8 and Q769J6. Structural detailsand sequence information on human ADAMTS13 can be found in Zheng et al.((2001) J. Biol. Chem. 276:41059-63).

The term “biologically active derivative or fragment thereof” as usedherein means any polypeptides with a biological function similar, orsubstantially similar, to that of ADAMTS13. The polypeptide sequences ofthe biologically active derivatives or fragments thereof may comprisedeletions, additions, and/or substitution of one or more amino acidswhose absence, presence, and/or substitution, respectively, do not haveany substantial negative impact on one or more biological activities ofthe ADAMTS13 protein. For example, alternative splicing gives rise to a130 kDa species that is a biologically active fragment of thefull-length protein. The biological activity of said polypeptides may bemeasured by well-known methods, for example, methods testing theproteolytic activity of ADAMTS13 on von Willebrand Factor (vWF), and/orsubsequent reduction and/or delay in downstream effects. By “downstreameffects” is meant one or more biological, biochemical, or physiologicalmanifestations of the action of ADAMTS13 protein on its nativesubstrate(s), whether the effect is a direct cause of ADAMTS13 function,or an indirect cause thereof, e.g., an effect resulting from a cascadeof events following ADAMTS13 activity. Assays include, withoutlimitation, methods testing the reduction and/or delay of plateletadhesion to the endothelium, the reduction and/or delay of plateletaggregation, the reduction and/or delay of the formation of plateletstrings, the reduction and/or delay of thrombus formation, the reductionand/or delay of thrombus growth, the reduction and/or delay of vesselocclusion, the proteolytical cleavage of vWF (e.g., FRETS-VWF73(Peptides International, Louisville, Ky.)), and/or the disintegration ofthrombi (see, e.g., U.S. Pat. No. 7,270,976, entitled “Methods formeasuring ADAMTS13 activity and protein on platelets and in plasma,”col. 6, line 55 to col. 10, line 34, and col. 12, line 1 to col. 18,line 25 and U.S. Pat. No. 7,468,258, entitled “Self-quenchinghomofluorophore compositions for detecting enzyme activity” col. 11,line 26 to col. 16, line 50; see also U.S. Patent Publication Nos.20070015703, entitled “ADAMTS13-containing compositions havingthrombolytic activity” at paragraphs [0036], [0043]-[0045], and [0053],and 20070065895, entitled “Substrates specific to von willebrand factorcleaving protease and method of assaying the activity”; and EuropeanApplication No. 1990421A1, entitled “Method for Detection of Conditionin Consciousness Disorder Patient and Kit for the Detection”, which areincorporated herein by reference with respect to assays for ADAMTS13polypeptides and derivatives and/or fragments thereof).

Recombinant ADAMTS13, e.g., recombinant human ADAMTS13, may be expressedby any method known in the art. One specific example is disclosed in WO02/42441, which is incorporated herein by reference with respect to themethod of preparing a recombinant ADAMTS13 nucleotide sequence (see page14, line 6 to page 18, line 4). In some embodiments, recombinantADAMTS13 is produced according to the following process: (i) preparing arecombinant ADAMTS13 nucleotide sequence by genetic engineering, e.g.via reverse transcription of RNA and/or amplification of DNA; (ii)introducing the recombinant ADAMTS13 nucleotide sequence into eukaryoticcells, e.g., by transfection, such as via electroporation ormicroinjection; (iii) cultivating the transformed cells, e.g., in acontinuous or batch-wise manner; (iv) allowing the expression ofrecombinant ADAMTS13, e.g., constitutively or upon induction; and (v)isolating samples comprising the expressed recombinant ADAMTS13, e.g.,from the culture medium or by harvesting the transformed cells; and (vi)purifying the ADAMTS13 protein from the sample, according to methodsdisclosed herein.

Recombinant ADAMTS13 may be produced by expression in a suitable hostsystem, preferably an eukaryotic host system, and more preferably asystem characterized in that it can produce a pharmacologicallyeffective ADAMTS13 molecule. Examples of eukaryotic cells include,without limitation, mammalian cells, such as CHO, COS, HEK 293, BHK,SK-Hep, and HepG2. In a preferred embodiment, CHO cells are used and thecells secrete recombinant ADAMTS13 protein into the culture medium.There is no particular limitation to the reagents or conditions used forrecombinantly expressing ADAMTS13 and any system known in the art orcommercially available may be employed.

“Sample” as used herein refers to any composition comprising ADAMTS13protein and non-ADAMTS13 impurities. A skilled artisan will recognizethat sample as used herein may be the result of producing recombinantADAMTS13 as described above. Accordingly, the sample may comprisesupernatant collected from culturing transformed cells, which expressrecombinant ADAMTS13; buffers comprising ADAMTS13 at one or more stepsof a process of purifying recombinant ADAMTS13 protein from culturemedium; and/or transformed cells harvested from cell culture.Alternatively, the sample may be blood, plasma, or a fraction of bloodor plasma.

In some embodiments, ADAMTS13 is purified from a sample comprising 100 Lcell culture supernatant. However, a skilled artisan will recognize thatthe methods of the invention may be scaled up as appropriate, e.g., forlarge scale production. Accordingly, in some embodiments, the methodcomprises purifying ADAMTS13 protein on a commercial production scale,e.g., from an at least about 250 L sample, an at least about 500 Lsample, or an at least about 1,000 L sample.

“Non-ADAMTS13 impurities” as used herein generally refers toprocess-related impurities. Impurities may include, e.g., host cellimpurities (such as contaminating host cell proteins, also referred toas host cell antigens) and other biomolecular impurities such as DNA,RNA, and cell debris; media component(s); solvents; detergents; and thelike. Additionally, non-ADAMTS13 impurities also include product-relatedimpurities, e.g., derivatives or fragments of ADAMTS13 protein, whichare not biologically active, or aggregates of ADAMTS13 protein. In thecase of blood or plasma, non-ADAMTS13 impurities may include otherproteins normally found in blood or plasma, e.g., albumin,immunoglobulins, etc. As used herein “aggregates” refers to structurescomprising more than one ADAMTS13 polypeptide molecule, or more than oneof any other protein molecule, which corresponds to high molecularweight structures or oligomeric structures, such as dimmers, trimers,and other multimers of the macromolecule. “Non-ADAMTS13 impurities” mayalso include virus contaminants. “Virus contaminants” refers to anyimpurities resulting from and/or derived from a virus, including, e.g.,virus particles, virus proteins, viral DNA, viral RNA, or fragmentsthereof.

The terms “purifying,” “purified,” “to purify” and the like refer toremoving, isolating, or separating ADAMTS13 from non-ADAMTS13impurities. For example, recombinant ADAMTS13 protein expressed inplant, bacterial, yeast, or mammalian host cells may be purified by theremoval of non-ADAMTS13 impurities comprising, e.g., host cell proteins.The percent purity may refer to the percent of ADAMTS13 protein versushost cell protein (e.g., CHO protein). “Substantially purified”recombinant ADAMTS13, is at least about 60% free, preferably at leastabout 75% free, and more preferably at least about 90% free (or about95%, about 99%, or about 99.9% free) from non-ADAMTS13 impurities. Inparticular, “substantially purified” recombinant ADAMTS13 is at leastabout 60% free, preferably at least about 75% free, and more preferablyat least about 90% free (or about 95%, about 99%, or about 99.9% free)from host cell proteins. Host cell proteins can be detected, forexample, by immunochemical methods using polyclonal antisera, asdiscussed in more detail below.

The removal of contaminants may also result in enrichment of theADAMTS13 protein. “Enrichment,” “enriching,” and “to enrich” as usedherein refer to an increase in the percent of recombinant ADAMTS13 inthe sample. Accordingly, enrichment of ADAMTS13 protein occurs when thepercent of ADAMTS13 is increased in a sample after some manipulation ofthe sample, e.g., subjecting the sample to one or more chromatographicsteps. In one embodiment, ADAMTS13 is sufficiently enriched when thereis at least about 10-fold reduction to about 115-fold reduction ofnon-ADAMTS13 impurities, particularly host cell proteins. In oneembodiment, ADAMTS13 is sufficiently enriched when there is at leastabout 20-fold (e.g., about 30-fold, about 40-fold, about 50-fold, about60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold,etc.) reduction of non-ADAMTS13 impurities, and in particular, thereduction occurs with respect to host cell proteins.

A skilled artisan will be able to use methods available in the art todetermine the fold reduction of non-ADAMTS13 impurities, particularlyhost cell proteins. For example, an assay for non-ADAMTS13 impuritiesmay be utilized. In one embodiment, the sample is conditionedsupernatant collected from the cultivation of transformed cellsexpressing recombinant ADAMTS13 and the assay to determine foldreduction of non-ADAMTS13 impurities measures levels of host cellproteins. In one particular embodiment, the transformed cells aretransformed CHO cells, and the assay is an enzyme-linked-immunosorbentserologic assay that measures CHO proteins. Fold reduction ofnon-ADAMTS13 impurities may be calculated, e.g., as the amount ofnon-ADAMTS13 impurities in the sample over the amount of non-ADAMTS13impurities eluted, as the impurity level in a load (e.g., in ppm)divided by the impurity level in the eluate (e.g., in ppm).

Host cell proteins may be detected, e.g., by immunochemical methodsusing polyclonal antisera against protein components of the host celland/or recombinant vector system used to manufacture ADAMTS13.Generally, antisera may be raised against antigen derived from the hostcell, wherein the host cell comprises an expression vector that is usedin the manufacturing process but that lacks the gene coding forADAMTS13. Host cell impurities may be extracted, using the method(s)identical and/or substantially similar to those described herein.Purified (or partly purified) host cell antigens obtained using themethod(s) identical and/or substantially similar to those describedherein may then be used for the preparation of antisera against proteincomponents of the host cell and recombinant vector system used tomanufacture ADAMTS13. The host cell proteins can be detected using theantisera in an immunoassay, for example, in an ELISA or by western blotanalysis. Host cell protein impurities may also be detected byseparating the sample to be analyzed by 2D gel electrophoresis andsilver staining and/or colloidal gold staining to detect proteinspresent. HPLC may also be used to quantify the levels of host cellimpurities; however, HPLC methods are not as sensitive as theimmunoassay or silver staining methods. Preferably, host cell impuritiesare reduced to below detectable levels using, e.g., one or more of theseanalytical methods.

As used herein, the term “about” denotes an approximate range of plus orminus 10% form a specified value.

Enrichment of ADAMTS13

Generally, the invention provides a method of purifying recombinantADAMTS13 protein (preferably human ADAMTS13 protein) from a samplecomprising ADAMTS13 protein and non-ADAMTS13 impurities, wherein themethod comprises chromatographically contacting the sample with (i)hydroxyapatite or (ii) hydroxyapatite and a mixed mode cationexchange/hydrophobic interaction resin in tandem, so as to enrich theamount of ADAMTS13 in the sample. “Chromatographically contacting” asused herein refers to contacting a sample or other mixture to beseparated with a chromatographic resin using any mode of chromatographydescribed herein and/or known in the art. Modes include, withoutlimitation, batch-mode and column chromatography. The contacting iseffected by exposing and/or incubating the sample on, in or within theresin, filtering the sample through the resin, or by any other means.The buffer used for chromatography often is a phosphate buffer.

In some embodiments, the sample is chromatographically contacted withhydroxyapatite under conditions that allow the ADAMTS13 protein toappear in the eluate from the hydroxyapatite. By “under conditions”refers to one or more parameters or variables under which thechromatography is carried out, including, e.g., column height, packing,buffer (pH, salt concentration, ionic strength, etc), temperature,pressure, and the like. That is, the sample is subjected tochromatography with hydroxyapatite under conditions that allow ADAMTS13protein, preferably a substantial portion of the ADAMTS13 protein in thesample, to not bind to the hydroxyapatide. If column chromatography isused, the ADAMTS13 protein, preferably a substantial portion thereof,will flow through the column, thereby enriching for ADAMTS13 in buffercoming off the column, as the flow-through fraction or eluate, whilenon-ADAMTS13 impurities are retained. If batch chromatography is used,the supernatant or supernatant fraction will comprise the ADAMTS13protein, or a substantial portion thereof “Eluate” is usedinterchangeably herein with “flow through”, “flow through fraction”,“supernatant”, or “supernatant fraction.” The eluate (or supernatant)can be collected. Such collection occurs, e.g., by centrifugation,sedimentation, filtration, etc. of the chromatographic resin after thesample is exposed to the resin and incubation completed. The eluate (orsupernatant) collected from the hydroxyapatite may be further subjectedto one or more steps according to the invention.

In some embodiments, for example, the method further compriseschromatographically contacting the eluate from the hydroxyapatite with amixed mode resin, such as a cation exchange/hydrophobic interactionresin, which binds the ADAMTS13 protein. That is, the ADAMTS13 proteinsample may be subjected to tandem chromatography, first withhydroxyapatite, preferably under conditions where a substantial portionof ADAMTS13 protein does not bind the hydroxyapatite, followed bychromatography with a mixed mode cation exchange/hydrophobic interactionresin that binds the ADAMTS13 protein. Additional details regarding thehydroxyapaptite chromatography step, and the optional tandem step ofmixed mode chromatography, using a cation exchange/hydrophobicinteraction resin, are provided below.

(a) Hydroxyapatite Chromatography

The hydroxyapatite chromatography step involves any method ofchromatography with hydroxyapatite, as described herein, as known in theart, or as can be appreciated by one of skill in the art, especially inlight of disclosures herein. Methods of chromatography withhydroxyapatite are well-known in the art. Hydroxyapatite has a chemicalformula of Ca₁₀(PO₄)₆ (OH)₂ and is a major constituent of bone and toothmineral, as well as other biological structures. Hydroxyapatite may beobtained from such natural sources or may be synthesized by well-knownmethods. Hydroxyapatite is widely used as a chromatographic medium orsupport, particularly for chromatographic separations of proteins. Theparticle size generally is not critical and may vary widely. Typicalparticle sizes range from about 1 μm to about 1,000 μm in diameter,preferably from about 10 μm to about 100 μm in diameter. The porositymay also vary widely. In preferred embodiments, the average porediameter ranges from about 100 Å to about 10,000 Å, more preferably fromabout 500 Å to about 3,000 Å, even more preferably 500 Å to 3,000 Å.

Various hydroxyapatite chromatographic media are available commercially,and any available form of the material can be used in the practice ofthe methods disclosed herein. Non-limiting examples of commerciallyavailable ceramic hydroxyapatite material that may be used includeMACRO-PREP™, Hydroxyapatite Types I and II (Biorad, Hercules, Calif.),and HA ULTROGEL® (PALL, Ann Arbor, Mich.). In one embodiment, the sampleis subject to chromatography with Hydroxyapatite type II (Biorad,Hercules, Calif.).

Surprisingly, it was discovered that upon chromatographically contactinga sample with hydroxyapatite, a significant or substantial portion ofnon-ADAMTS13 impurities in the sample bind hydroxyapatite, while asignificant or substantial portion of the ADAMTS13 protein remains insolution. Accordingly, as discussed above, treatment of the sample withhydroxyapatite may be performed in batch-mode or in columnchromatography mode according to well-known methods, and sufficientlyenriched ADAMTS13 protein collected in the supernatant or in the eluate,respectively.

As used herein, “substantial portion” refers to a recovery yield in thesupernatant or eluate of about 30% to about 100% (e.g., about 40% toabout 90%, e.g., about 50% to about 80%, e.g., about 60% to about 70%)of recombinant ADAMTS13 protein from the sample compared to that priorto the hydroxyapatite chromatography step. For example, recovery yieldin the supernatant or eluate of about 50% to about 100% indicates thatthe sample was subject to chromatography with hydroxyapatite underconditions that allow a substantial portion of ADAMTS13 protein to flowthrough.

In preferred embodiments, the sample to be subjected to hydroxyapatitechromatography has a low conductivity, e.g., between about 3 mS/cm andabout 15 mS/cm at room temperature, preferably less than about 10 mS/cmat room temperature. In one embodiment, the sample has a conductivity of6 mS/cm at room temperature. In another embodiment, the sample has aconductivity of 7 mS/cm at room temperature. A skilled artisan willreadily appreciate that conductivity of the sample may be adjusted witha salt solution comprising neutral salts, e.g., sodium chloride,potassium chloride, sodium sulfate, sodium phosphate, potassiumphosphate, and the like, and can be suitably buffered with about 20 mMphosphate buffer. The sample preferably has a pH between about 6.5 andabout 9.0, and preferably, has a pH between 7 and 8. The sample mayremain in contact with the hydroxyapatite for any length of time thatwill allow sufficient binding of non-ADAMTS13 impurities, e.g., forabout 5 minutes to about 24 hours. Enriched ADAMTS13 may be collected inthe supernatant fraction or the flow through fraction, which may includeeluate from subsequent washes, particularly the first wash.

In one embodiment, subjecting the sample to chromatography withhydroxyapatite under conditions that allow a substantial portion ofADAMTS13 protein to remain in the supernatant or eluate results inenriched ADAMTS13, e.g., about 10-fold reduction to about 115-foldreduction of non-ADAMTS13 impurities, particularly host cell proteins,compared to the sample prior to chromatography with hydroxyapatite. Inone embodiment, chromatography with hydroxyapatite reduces host cellproteins in the sample by at least about 20-fold (e.g., about 30-fold,about 40-fold, about 50-fold, about 60-fold, about 70-fold, about80-fold, about 90-fold, e.g., about 100-fold, etc.).

In preferred embodiments, subjecting the sample to chromatography withhydroxyapatite under conditions that allow a substantial portion ofADAMTS13 protein to remain in the supernatant or eluate results in about90% to about 99% removal of non-ADAMTS13 impurities, particularly hostcell proteins. In one embodiment, subjecting the sample tochromatography with hydroxyapatite results in at least about 90% (e.g.,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, about 99.5%, etc.) removal of non-ADAMTS13impurities, particularly removal of host cell proteins. Accordingly,methods disclosed herein comprising enriching for ADAMTS13 protein bysubjecting the sample to chromatography with hydroxyapatite underconditions that allow a substantial portion of the ADAMTS13 protein toremain in the supernatant or eluate may also provide a buffer comprisingADAMTS13 protein that is substantially purified.

Exemplary column conditions that allow a substantial portion of ADAMTS13to flow through a hydroxyapatite chromatography column are provided inthe examples below. Generally, to allow a substantial portion ofADAMTS13 protein to flow through during chromatography withhydroxyapatite, the chromatography column preferably will have a bedheight between about 5 cm to about 30 cm, e.g., 20 cm to 30 cm.Additionally, prior to subjecting the sample to chromatography withhydroxyapatite, e.g., before loading the sample onto the hydroxyapatitecolumn, the column may first be washed, activated, and/or equilibratedrespectively with well-known wash, activation, and/or equilibrationbuffers, particularly those suggested by the manufacturer of thehydroxyapatite. In one embodiment, the column is activated andequilibrated with the same buffer, e.g., a buffer comprising 20 mM Na/KPO₄, having a pH of 7.0 and having a conductivity of 5.5 mS/cm at roomtemperature.

(b) Mixed Mode Cation Exchange/Hydrophobic Interaction Chromatography

In one embodiment, ADAMTS13 protein is enriched by tandem chromatographywith the hydroxyapatite followed by a mixed mode cationexchange/hydrophobic interaction resin that binds ADAMTS13 protein. Ithas been surprisingly discovered that ADAMTS13 binds the mixed modecation exchange/hydrophobic interaction resin while non-ADAMTS13impurities either remain in solution or bind much more strongly to themixed mode cation exchange/hydrophobic interaction resin. Accordingly,treatment of the sample with hydroxyapatite followed by treatment with amixed mode cation exchange/hydrophobic interaction resin may beperformed in successive batch-mode or in successive columnchromatography mode according to well-known methods and sufficientlyenriched ADAMTS13 protein may be collected in the final supernatantfraction or final eluate pool after subjecting the sample to tandembatch-mode chromatography or tandem column chromatography, respectively.Accordingly, as described herein, ADAMTS13 protein is enriched bysubjecting the sample to chromatography with hydroxyapatite underconditions that allow a substantial portion of ADAMTS13 protein toremain in the supernatant or to flow through a column comprisinghydroxyapatite as the eluate. After treatment with hydroxyapatite, thecollected supernatant or eluate comprising enriched ADAMTS13 optionallyis subjected to batch-mode or column chromatography with a mixed modecation exchange/hydrophobic interaction resin. In one embodiment, thesupernatant or eluate from the hydroxyapatite step is fed into achromatography column comprising the mixed mode cationexchange/hydrophobic interaction resin. Batch-mode or columnchromatography with a mixed mode cation exchange/hydrophobic interactionresin can be carried out by any method described herein, known in theart, or as can be appreciated by one of skill in the art, especially inlight of disclosures herein. A preferred mixed mode cationexchange/hydrophobic interaction resin, suitable for use afterchromatography with hydroxyapatite, is sepharose-based matrix comprisinga hydrophilic linker. The hydrophilic linker may comprise a functionalligand, e.g., via a thio-ether group. The hydrophilic ligand may benegatively-charged and may further comprise a hydrophobic group, e.g., ahydrocarbon. A hydrophilic ligand further comprising a hydrophobic groupcan create a mixed mode ligand, that is, a ligand with multimodalfunctionality, suitable for performing mixed mode chromatography, asdescribed herein. In some embodiments, the ionic capacity of the mixedmode cation exchange/hydrophobic interaction resin may be between about0.07 mM/mL to about 0.09 mM/mL and have a pH stability between about 2to about 14. Generally, ADAMTS13 will bind the mixed mode cationexchange/hydrophobic interaction resin via ionic, hydrogen, and/orhydrophobic bonds.

Examples of commercially available mixed mode cationexchange/hydrophobic interaction resins that may be used in accordancewith the methods described herein include, without limitation, CAPTO™MMC medium (GE Healthcare) and SampliQ SAX (Agilent Technologies, SantaClara, Calif.). In a preferred embodiment, the mixed mode cationexchange/hydrophobic interaction resin is CAPTO™ MMC. CAPTO™ MMC is amultimodal weak cation exchanger based on rigid, highly cross-linked,beaded agarose with a mean particle size of about 75 μm. It comprisesligands with multimodal functionality that bind proteins at high saltconcentrations. It has a typical flow velocity of about 600 cm/h for anabout 1 m diameter column, with about 10 cm to about 20 cm bed height atabout 20° C., using process buffers with the about same viscosity aswater at less than about 3 bar (about 0.3 MPa).

During chromatography with a mixed mode cation exchange/hydrophobicinteraction resin, ADAMTS13 binds to the mixed mode cationexchange/hydrophobic interaction resin and is further isolated fromnon-ADAMTS13 impurities (e.g., host-cell proteins present in the samplepre-enrichment). Where the mixed mode chromatography step is performedon a column, the cation exchange/hydrophobic interaction resin absorbsADAMTS13 protein, while contaminating non-ADAMTS13 impurities areremoved from the process stream and separated from the ADAMTS13 proteinin the sample by flowing through the chromatography column.

The mixed mode cation exchange/hydrophobic interaction resin to whichADAMTS13 is adsorbed is then washed, e.g., to remove loosely-boundcontaminants or impurities and/or to adjust buffer conductivity inpreparation for elution of ADAMTS13 from the resin. That is, after thesample is chromatographically contacted with hydroxyapatite, and thecollected supernatant or eluate comprising ADAMTS13 ischromatographically contacted with mixed mode cation/hydrophobicinteraction resin and adsorbed thereto, the mixed modecation/hydrophobic interaction resin is washed with wash buffer.Generally, the wash buffer will comprise a buffering ion phosphate and aneutral salt and will have a high pH such that binding of ADAMTS13 tothe mixed mode cation/hydrophobic interaction resin is weakened asrelevant parameters of the buffer are increased, e.g., with increasingsalt concentration and/or pH of the buffer. In one embodiment, theADAMTS13-bound mixed mode cation/hydrophobic interaction resin is firstwashed with an equilibration buffer, e.g., an equilibration buffercomprising about 20 mM phosphate and about 25 mM NaCl and having a pH ofabout 7.0 at room temperature. Subsequent washes may be performed with awash buffer, comprising, e.g., about 20 mM phosphate, about 80 mM NaCland having a pH of about 8.0 at room temperature. The ADAMTS13-boundmixed mode cation/hydrophobic interaction resin may be subject to afinal wash with a buffer comprising, e.g., 50 mM Na/K PO₄ and 160 mMNaCl, and having a pH of 8.0, and a conductivity of 16.5 mS/cm at roomtemperature.

After hydroxyapatite chromatography, followed by mixed modechromatography with a cation exchange/hydrophobic interaction resin thatbinds ADAMTS13 protein, and optional washing, recombinant ADAMTS13protein is eluted from the mixed mode chromatography resin with anelution buffer. Generally, the elution buffer will comprise about 5 mMto about 100 mM buffering ions, e.g., 20 mM to 50 mM buffering ions.Exemplary buffers include, but are not limited to, phosphate, tris,HEPES, imidazole, histidine, MES, citrate, Gly-Gly, tris/acetate, andthe like. The elution buffer also generally comprises monovalent ordivalent cations, such as, but not limited to, sodium, potassium, orcalcium ions, preferably at high concentrations in salt form (e.g., withCl, PO₄, SO₄, or OAc anions, and the like.). In a preferred embodiment,the elution buffer comprises sodium ions at high concentration, e.g., atgreater than about 700 mM Na⁺. The elution buffer may have a pH rangingfrom about 7 to about 11. In one embodiment, the elution buffercomprises 50 mM Na/K PO₄, 1,000 mM NaCl and has a pH of 8.0 and aconductivity of 93 mS/cm at room temperature.

Exemplary conditions that allow enriched ADAMTS13 obtained from ahydroxyapatite column to bind to a mixed mode cationexchange/hydrophobic interaction resin in a column and to be isolatedfrom non-ADAMTS13 impurities are provided in the examples below.Generally, the bed height of the mixed mode cation exchange/hydrophobicchromatography column may be about 1 cm to about 100 cm, or even higherdepending on the sample volume. Further, the ratio of the hydroxyapatitechromatography column volume to the column volume of the mixed modecation exchange/hydrophobic interaction chromatography column may beabout 10:1, depending, e.g., on amounts of non-ADAMTS13 impurities inthe sample compared to the amount of ADAMTS13.

Additionally, a skilled artisan will recognize that, for embodimentscomprising tandem chromatography with hydroxyapatite and a mixed modecation exchange/hydrophobic interaction resin, the resins and buffersused for washing, activation, and/or equilibrating will in certainembodiments be selected to be compatible with both columns. In oneembodiment, the columns are activated separately. In another embodiment,the columns are equilibrated, loaded, and washed once in tandem,followed by one or more second or subsequent wash(es) and elution(s)applied only to the mixed mode cation exchange/hydrophobic interactionresin. In one embodiment, the buffers for activating and equilibratingthe mixed mode cation exchange/hydrophobic interaction chromatographycolumn are the same as those used to activate and equilibrate thehydroxyapatite column. In one embodiment, the buffer for activating andequilibrating the mixed mode cation exchange/hydrophobic interactionchromatography column is a buffer comprising 20 mM Na/K PO₄ and having apH of 7.0 and a conductivity of 5.5 mS/cm at room temperature.

In preferred embodiments, the sample is subjected to tandemchromatography with hydroxyapatite and mixed mode cationexchange/hydrophobic interaction resins under conditions that allow asubstantial portion of ADAMTS13 protein to flow through thehydroxyapatite resin, followed by being bound and then eluted from themixed mode cation exchange/hydrophobic interaction resin. In morepreferred embodiments, this tandem chromatography results insufficiently enriched ADAMTS13. In some embodiments, subjecting asample, e.g., conditioned supernatant collected from the cultivation oftransformed host cells expressing recombinant ADAMTS13, which may bepre-enriched, to the tandem chromatography described herein yields about40% to about 80% ADAMTS13 (e.g., between about 45% to about 75%, e.g.,between about 50% to about 70%, e.g., about 55% to about 65%) and/orabout 40% to about 90% activity (e.g., between about 45% to about 85%,e.g., between about 50% to about 80%, e.g., about 55% to about 75%).

In some embodiments, the tandem chromatography removes about 90% toabout 99% host cell impurities. In one embodiment, tandem chromatographyas described herein increases the purity of ADAMTS13 by at least about600 fold, e.g., by at least about 650-fold, e.g., by at least about700-fold, e.g., by at least about 800-fold, e.g., by at least about900-fold, e.g., by at least about 1,000-fold, e.g., by at least about1,100-fold, e.g., by at least about 1,200-fold, e.g., by at least about1,300-fold, e.g., by at least about 1,400-fold, or by at least about1,500-fold compared to the purity of ADAMTS13 prior to subjecting thesample to tandem chromatography.

In some embodiments, subjecting a sample, e.g., conditioned supernatantcollected from the cultivation of transformed host cells expressingrecombinant ADAMTS13, which may be pre-enriched, preferably by UF/DFand/or anion exchange, to the tandem chromatography described hereinresults in a sample with about 600 to about 1,500 ppm non-ADAMTS13impurities (e.g., host cell antigens). For example, the tandemchromatography may result in a sample with about 750 to about 1250 ppmnon-ADAMTS13 impurities, preferably a sample with less than about 1,000ppm non-ADAMTS13 impurities.

In some embodiments, the tandem chromatography results in an about1,000-fold reduction to an about 3,000-fold reduction of non-ADAMTS13impurities (in particular, host cell antigens) compared to the sampleprior to the tandem chromatography. In some embodiments, subjectingconditioned supernatant collected from the cultivation of transformedhost cells expressing recombinant ADAMTS13, which may be pre-enriched,to the tandem chromatography described herein reduces non-ADAMTS13impurities (e.g., host cell antigens) by at least about 1,000-fold,e.g., by at least about 1,300-fold, e.g., by at least about 1,500-fold,e.g., by at least about 2,000-fold, e.g., by at least about 2,500-fold,e.g., by at least about 3,000-fold, etc.

Accordingly, in preferred embodiments, the elution buffer from the mixedmode cation exchange/hydrophobic interaction resin, which comprisesrecombinant ADAMTS13, provides a composition comprising ADAMTS13 proteinthat is substantially purified.

Pre-Enrichment Preparation of the Sample

In some embodiments, the method disclosed herein further comprisespreparing the sample comprising ADAMTS13 for enrichment bychromatography with hydroxyapatite or tandem chromatography withhydroxyapatite and mixed mode cation exchange/hydrophobic interactionresin. In this optional pre-enrichment step, the ADAMTS13 in the samplemay be either or both (a) concentrated by ultrafiltration/diafiltration(UF/DF); and/or (b) chromatographically contacted with an ion exchangeresin, to which ADAMTS13 binds and from which it subsequently is eluted.

(a) Pre-Enrichment Ultrafiltration/Diafiltration (UF/DF)

In an optional pre-enrichment step, ADAMTS13 in a sample is concentratedby pre-enrichment ultrafiltration, and the buffer of the sampleexchanged by diafiltration. The pre-enrichmentultrafiltration/diafiltration step typically is performed prior toenrichment of ADAMTS13 by chromatography with hydroxyapatite or tandemchromatography with hydroxyapatite followed by a mixed mode cationexchange/hydrophobic interaction resin as described above. Thepre-enrichment ultrafiltration/diafiltration step typically is performedprior to any pre-enrichment anion exchange chromatography (ifperformed). This pre-enrichment ultrafiltration/diafiltration (UF/DF)step may be effective in removing small-molecular weight components,e.g., small-molecular weight components of the cell culture media. Suchcomponents may bind to a subsequent chromatography column and decreasethe capacity of the column for ADAMTS13. Accordingly, pre-enrichmentUF/DF can optimize loading for later chromatography steps. In oneembodiment, small-molecular weight components below about 30 kDa areremoved, or at least a substantial portion thereof. In some embodiments,small-molecular components removed (or substantially removed) arecomponents of below about 60 kDa, below about 55 kDa, below about 50kDa, below about 45 kDa, below about 40 kDa, below about 35 kDa, belowabout 30 kDa, below about 25 kDa, below about 20 kDa, etc.

The pre-enrichment ultrafiltration/diafiltration step also is used incertain embodiments to exchange ADAMTS13 into an appropriate buffersolution for subsequent processing and/or to further concentrate thesample. In one embodiment, the appropriate buffer solution is a lowconductivity buffer appropriate for pre-enrichment anion exchangechromatography, if such anion exchange chromatography is to beperformed. For example, the low conductivity buffer will have aconductivity of less than about 10 mS/cm, e.g., about 7 mS/cm to about 8mS/cm, e.g., 7 mS/cm at room temperature, and may have a pH equal to orgreater than about 7.0.

In another embodiment, the appropriate buffer solution is an enrichmentbuffer appropriate for enrichment by chromatography with hydroxyapatite,which may be followed with chromatography on a mixed mode cationexchange/hydrophobic interaction resin. For example, the enrichmentbuffer may comprise 20 mM Na/K PO₄ and have a pH of about 7 at roomtemperature. In another embodiment, the appropriate buffer solution alsocomprises calcium and/or zinc ions, either or both of which stabilizeADAMTS13 protein. In one embodiment, the appropriate buffer solutioncomprises calcium ions at a concentration of less than about 10 mM,e.g., 2 mM. In another embodiment, the appropriate buffer solution issupplemented with zinc ions at a concentration of less than about 50 μM,e.g., 5 μM.

In some embodiments, the appropriate buffer solution comprises abuffering agent that has buffering capacity in solutions with a pH equalto or greater than about 7.0. In one embodiment, the buffering agent isselected from the group consisting of phosphate, tris, HEPES, imidazole,histidine, MES, citrate, Gly-Gly, Tris/acetate, etc.

The sample obtained after this pre-enrichment UF/DF step may be used insubsequent purification steps, e.g., the sample may be a UF/DFconcentrated pool comprising host cell proteins to be removed bychromatography with hydroxyapatite, or hydroxyapatite chromatographyfollowed by mixed mode chromatography on a cation exchange/hydrophobicinteraction resin. In some embodiments, the sample following thepre-enrichment UF/DF step has been concentrated by about 10 fold toabout 20 fold, e.g., by about 15 fold, compared to the sample before thepre-enrichment UF/DF step.

(b) Pre-Enrichment Anion Exchange Chromatography

Another optional pre-enrichment step comprises pre-enrichmentchromatography, which may be performed prior to enrichment of ADAMTS13by chromatography with hydroxyapatite or tandem chromatography withhydroxyapatite followed by a mixed mode cation exchange/hydrophobicinteraction resin. A skilled artisan will recognize that thepre-enrichment chromatography may be performed after the optionalpre-enrichment ultrafiltration/diafiltration step. Alternatively, thepre-enrichment chromatography may be performed by itself, i.e., withoutthe optional pre-enrichment ultrafiltration/diafiltration step.

In some embodiments, the pre-enrichment chromatography step compriseschromatographically contacting the sample comprising ADAMTS13 with ananion exchange resin and eluting the ADAMTS13 protein from the anionexchange resin. That is, the ADAMTS13 is bound to an anion exchangeresin and subsequently eluted therefrom. As used herein, the term “anionexchange resin” refers to any resin suitable for anion exchangechromatography and that has a net positive charge, e.g., due to apositively-charged group (at neutral pH). Examples include, but are notlimited to, diethylaminoethane (DEAE), dimethylethanolamine (DMAE),polyethyleneimine (PEI), quaternary aminoethane (QAE),trimethylaminoethyl (TMAE), quarternary ammonium (Q), and the like, andcombinations thereof.

In one embodiment, the anion exchange resin also has one or more of thefollowing features: large pores, perfusion flow behavior, and convectiveflow behavior. Non-limiting examples of commercially available anionexchange resins that may be used in the pre-enrichment step disclosedherein include Q-Sepharose Fast Flow (GE Healthcare, Piscataway, N.J.),ANX-Sepharose Fast Flow low sub (GE Healthcare), DEAE-Sepharose FastFlow (GE Healthcare), DEAE-Toyopearl (Tosoh Bioscience LLC, Grove City,Ohio), QAE-Toyopearl (Tosoh Bioscience LLC), POROS® Q (AppliedBiosystems, Foster City, Calif.), POROS® 50D (Applied Biosystems),POROS® 50PI (Applied Biosystems), Convective Interaction Media (CIM®;BIA Separation), Fractogel-DMAE (Capitol Scientific Inc., Austin, Tex.),Fractogel EMD-TMAE (Capital Scientific Inc., Austin, Tex.), MatrexCellufine DEAE (Chisso Corp., Rye, N.Y.), and the like.

During pre-enrichment anion exchange chromatography, ADAMTS13 binds tothe anion exchange resin and is isolated from non-ADAMTS13 impurities(e.g., host-cell components that may be present in the pre-enrichmentUF/DF concentrated pool). Generally, the anion-exchange resin absorbsADAMTS13 protein, while non-ADAMTS13 impurities with isoelectric pointsgreater than the operating pH are removed from the process stream byflowing through the anion exchange column. Non-ADAMTS13 impurities withisoelectric points below the operating pH bind more strongly, preferablymuch more strongly, to the resin, such that they preferably do notco-elute with the ADAMTS13 protein. The column to which ADAMTS13 isadsorbed is then washed prior to elution, e.g., to remove loosely-boundimpurities or contaminants and/or to adjust the conductivity of thebuffer in preparation for elution. Typically, bound ADAMTS13 is elutedfrom the anion exchange resin by increasing the ionic strength of thebuffer. In one embodiment, ADAMTS13 is eluted by step elution.Generally, the loaded sample and wash buffer have a pH of between about7 to about 9, e.g., 7.7, and a conductivity of less than about 10 mS/cm(e.g., 6.5 mS/cm) at room temperature. The elution buffer(s) may have apH of about 6 to about 9 (e.g., 7) and have a conductivity of greaterthan about 10 mS/cm (e.g., 16.5 mS/cm) at room temperature.

Typically, the eluate from the anion exchange chromatography step yieldsabout 60% to about 120% ADAMTS13 activity (e.g., yields about 70% orabout 80% to about 107% ADAMTS activity) and/or comprises recombinantADAMTS13 with a purity of about 20% to about 70%, (e.g., a purity ofabout 30%, about 40%, about 50%, about 60%, etc). In one embodiment,anion exchange chromatography reduces non-ADAMTS13 impurities by about2-fold to about 5-fold. In a preferred embodiment, the percent yieldafter pre-enrichment preparation can be about 75%.

The eluted ADAMTS13 may then be enriched by subjecting the sample tochromatography with hydroxyapatite that allows a substantial portion ofADAMTS13 protein to flow through or subjecting the sample to tandemchromatography with hydroxyapatite under conditions that allow asubstantial portion of ADAMTS13 protein to flow through, followed bychromatography with a mixed mode cation exchange/hydrophobic interactionresin that binds ADAMTS13 protein, as described above.

Virus Inactivation

A skilled artisan will recognize that methods of virus inactivation maybe particularly useful in purifying recombinant ADAMTS13 from samplesthat comprise or potentially comprise virus contaminants (impuritiesresulting from and/or derived from viruses, including, e.g., virusparticles, virus protein, viral DNA, viral RNA, and fragments thereof).Accordingly, in one embodiment, the method disclosed herein furthercomprises at least one virus inactivation step. The term “virusinactivation” refers to either or both the situation wherein viruses aremaintained in the solution but are deactivated or inactivated (e.g.,rendered non-viable, for example, by dissolving the lipid coat oflipid-enveloped viruses); and to the physical removal of the virusesand/or virus contaminants from the sample (for example, by sizeexclusion). Thus, in the context of the disclosure herein, “virusinactivation” refers to either or both viral deactivation and viralremoval.

If performed, virus inactivation may occur once or more than oncethroughout the entire purification process. Additionally, it may occurprior or subsequent to subjecting the sample to chromatography withhydroxyapatite. In some embodiments, virus inactivation occurs prior andsubsequent to the optional step of polishing by cation exchangechromatography, described in more detail below. However, a skilledartisan will recognize that virus inactivation may optionally occur, ifat all, at any step during the purification process. Further, a skilledartisan can recognize the appropriate timing for virus inactivation.

Methods of rendering lipid-enveloped viruses non-viable are well-knownin the art. Generally, methods of deactivating (or inactivating)lipid-enveloped viruses in a sample comprise adding a solvent-detergentmixture to the sample (see, e.g., Edwards, et al. (1987) “Tri(n-butyl)phosphate/detergent treatment of licensed therapeutic and experimentalblood derivatives” Vox Sang 52: 53-59 (see especially pages 54-55); andU.S. Pat. No. 4,540,573 (col. 7, line 9 to col. 12, line 42); U.S. Pat.No. 4,764,369 (col. 7, line 17 to col. 12, line 47); U.S. Pat. No.4,939,176 (col. 3, line 59 to col. 10, line 14); U.S. Pat. No. 5,151,499(col. 2, line 59 to col. 11, line 38); U.S. Pat. No. 6,090,599 (col. 4,line 20 to col. 8, line 67); U.S. Pat. No. 6,468,733 (col. 5, line 12 tocol. 9, line 36); and U.S. Pat. No. 6,881,573 (col. 5, line 63 to col.14, line 9); each of which is incorporated herein by reference). Thesolvent-detergent combination used to deactivate lipid-coated virusesmay be any solvent-detergent combination known in the art and preferablycomprises a non-ionic detergent and an organic solvent. Non-limitingexamples include Tri-N-butyl phosphate (TnBP) and TRITON X-100™, as wellas TWEEN 80™ (CAS 9005-65-6), polyoxyethylene sorbitan monooleate,sodium cholate, and the like. The concentration of the solvent(s) and/ordetergent(s) may be those commonly used in the art, for example, greaterthan about 0.1% TnBP and greater than about 0.1% TRITON X-100™.

In some embodiments, the conditions under which the solvent-detergentmixture inactivates the viruses comprise about 10 to about 100 mg/ml ofsolvent-detergent, at a pH level ranging from about 5 to about 8, and atemperature ranging from about 2° C. to about 37° C., preferably fromabout 12° C. to about 25° C., for about 30 minutes to about 24 hours,preferably about 30 minutes to about 1 hour. In some embodiments, themixture is slightly shaken or stirred during the treatment. In oneembodiment, the virus inactivation step comprises adding asolvent-detergent mixture (e.g., as solvent-detergent mixture comprising0.3% TnBP, 1% TRITON X-100™, and 0.3% TWEEN 80™) to the sample for atleast 1 hour, at 15° C. to 25° C. In another embodiment, the sample istreated with a solvent-detergent mixture comprising 0.3% TnBP, 1% TRITONX-100™, and 0.3% TWEEN 80™ for 30 minutes at 12° C. to 16° C. Othersolvent-detergent combinations and/or suitable conditions may be used,as will be apparent to one versed in the art, such as combinations ofpolysorbate or cholate and tri-n-butyl phosphate. Such combinations mayrequire longer treatment times, e.g., 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, or more.

Inactivation can be brought about by any means known in the art. Forexample, inactivation can be stopped by dilution, preferably by dilutionwith cold dilution buffer. For example, in some embodiments,inactivation is stopped by dilution with one volume of cold dilutionbuffer comprising about 20 mM MES, and having a pH of about 6 at roomtemperature.

After deactivating lipid-coated viruses with the solvent-detergentcombination, the solvent-detergent mixture may be removed. For example,the solvent-detergent mixture may be removed via chromatography or othersuitable means. In some embodiments, chromatography with asolvent-detergent removal (SDR) resin is used, such as, e.g., HyperD™resin (Biosepra Inc., MA) (see, e.g., U.S. Pat. No. 6,468,733 (col. 5,line 12 to col. 9, line 36), incorporated herein in its entirety byreference).

Inactivating Virus Contaminants with Immobilized Protein

In some embodiments, virus inactivation comprises viral deactivationwith solvent-detergent while the protein is immobilized. Such aprocedure may be used in virus inactivation of the ADAMTS13 polypeptidedescribed herein, as well as for other proteins. Other proteins mayinclude, without limitation, any protein or biologic from a source thatmay have viral contaminants, including immune system proteins(antibodies, monoclonal antibodies, fusion proteins, Fc fusions, majorhistocompatibility antigens, T cell receptor), enzymes (oxidoreductases,transferases, hydrolases, lyases, isomerases, ligases), structuralproteins, fibrous proteins (such as cytoskeletal proteins, like actin,Arp2/3, coronin, dystrophin, keratin, myosin, spectrin, Tau protein,tubulin and extracellular matrix proteins, like collagen, elastin,F-spondin), globular proteins, plasma proteins (serum albumin and serumamyloid P component), coagulation factors (like complement proteins,Factor VIII, Factor XIII, fibrin, protein C, protein S, protein Z,protein Z-related protease inhibitor, thrombin, von Willebrand Factor),C-reactive protein, hemoproteins, cell adhesion proteins (cadherin,ependymin, integrin, NCAM, selectin), transmembrane transport proteins(CFTR, glycophorin D, scramblase), ion channels (acetylcholine receptorpotassium channel), synport/antiport proteins (glucose transporter),hormones and growth factors (epidermal growth factor insulin,insulin-like growth factor, oxytocin), receptors (transmembranereceptors, G-protein-coupled receptor, rhodopsin, intracellularreceptors like estrogen receptor), DNA-binding proteins (histones),transcription regulation proteins (c-myc FOXP2, FOXP3, MyoD, p53),nutrient storage/transport proteins (ferritin), chaperone proteins,macromolecular complexes (nucleosome, ribonucleoprotein, signalrecognition particle, spliceosome), and the like. In preferredembodiments, the protein is recombinant protein, particularly proteinssensitive to aggregation when exposed to organic solvents anddetergents. In some embodiments, the protein is ADAMTS13 protein, inparticular recombinant ADAMTS13, or a different protein (in particular,a different recombinant protein). In some embodiments, the recombinantprotein is a blood coagulation factor. In some embodiments, the proteinis, e.g., one or more of Factor VIII, Factor II, thrombin, Factor VIIa,Factor IX, von Willebrand factor, anti-MIF antibodies, and in particularproteins amenable to chromatographic purification and/or proteinssensitive to treatment with solvent-detergent. Accordingly, anotheraspect of the instant invention is directed at virus inactivation of animmobilized protein. Preferably, virus inactivation is carried out inconjunction with a protein purification procedure, such that theprocedure involves virus inactivation of a protein preparation byimmobilizing the protein being purified.

The conventional solvent-detergent virus inactivation step applied indownstream processes for purifying various proteins, such as thosedescribed above, generally involves adding the solvent-detergent mixturein solution to the sample being purified, e.g., in a batch procedure. Inthe batch procedure, a sample comprising the protein is treated with thesolvent-detergent mixture (e.g., a mixture comprising about 1% TritonX-100, about 0.3% tri-N-butyl phosphate, and about 0.3% Polysorbate 80)in a stirred vessel (e.g., a tank for large scale purifications). Afterdissolution of the solvent-detergent chemicals, the treated samplesolution can be pumped into a second stirred vessel, where by definitionthe actual virus inactivation takes place, as here the protein solutionis incubated to allow such deactivation to take place (e.g., for about30 minutes to about one hour).

In contrast, some embodiments of the instant invention involve virusinactivation of a composition comprising a protein, e.g., a proteinbeing purified, where the protein is immobilized. The process comprisescontacting the composition comprising the protein of interest with asolvent-detergent mixture while the protein is immobilized, rather thanthe target protein being in solution. In preferred embodiments, theprotein is immobilized on a chromatographic resin. Virus inactivationwhere the protein is immobilized on a chromatographic resin, e.g., on achromatographic column, is referred to herein as “on-column” virusinactivation. The purification of ADAMTS13 on Poros S, described in theExamples below, provides one embodiment of this process, where virusinactivation is carried out on-column. One of skill in the art willrecognize that the protein may be immobilized onto various supports, bya variety of means. For example, protein may be bound to any solid orsemi-solid support, including a glass slide, beads, matrix, ormembranes. Immobilization may result from any process whereby theprotein is fixed to the support relative to other components of theprotein solution. Immobilization may occur due to one or more types ofbonds between the groups on the support and groups on the protein, suchas, e.g., by covalent linkage, hydrogen bonds, electrostaticinteractions, van der Wasls forces, and the like, or combinationsthereof.

Virus inactivation of immobilized protein on a chromatographic columncan simplify purification. For example, rather than requiring more thanone vessel (such as a two tank system used in large scalepurifications), chromatographic purification and virus inactivation maybe carried out in the same vessel, e.g., on the same chromatographiccolumn. This simplifies the downstream processes of proteinpurification, e.g., reducing time, conserving reagents, and/orincreasing efficiency. In some embodiments, the chromatographic columnis a cation exchange resin. In some embodiments, the chromatographiccolumn is an anion exchange resin.

An additional and surprising benefit of certain embodiments of virusinactivation of immobilized protein is the reduction in aggregateformation. Some proteins show sensitivity towards solvent-detergentmixtures, e.g., forming aggregates when contacted with thesolvent-detergent reagents in solution. Without being limited to aparticular theory or hypothesis, contacting the sensitive protein withthe solvent-detergent mixture while it is immobilized, e.g., while theprotein is bound to a chromatographic resin, can prevent the formationof aggregates based simply on the physical inability of the immobilizedprotein molecules to contact each other. In some embodiments, theinactivation results in the formation of less than about 20% aggregates,less than about 18%, less than about 15%, less than about 12%, less thanabout 10%, or less than about 5% aggregates. And, in certainembodiments, the level of aggregation is reduced by at least about 10%,about 20%, about 50% or about 100% as compared to the level ofaggregation when the protein preparation is subjected to virusinactivation where the protein is not immobilized.

In one preferred embodiment, the protein is loaded onto achromatographic resin and the solvent-detergent treatment is used as awash step, preferably a wash step that continues for a long enoughincubation period to allow inactivation of lipid-enveloped viruses. Forexample, the wash step preferably continues for about 30 minutes toabout one hour. The solvent-detergent mixture will comprise non-ionicdetergent and organic solvent at concentrations suitable to effect suchvirus inactivation, as described above. For example, in someembodiments, the solvent-detergent mixture comprises 1% Triton X-100,0.3% tri-N-butyl phosphate, and 0.3% Polysorbate80. Additional detailsfor some particular embodiments are provided below, with respect toADAMTS13 purification.

Virus inactivation may also comprise viral removal, e.g., by filtration,such as nanofiltration using a nanofilter. Such viral removal may occuralone, or in combination with viral deactivation (inactivation), e.g.,the viral deactivation step comprising treatment with asolvent-detergent mixture as described above. When virus inactivationcomprises both viral deactivation and viral removal, viral removal mayoccur prior to and/or subsequent to the viral deactivation bysolvent-detergent treatment. Generally, viral removal from a sampleinvolves filtering the sample, e.g., passing the sample through a filterhaving a pore size that maintains ADAMTS13 in the sample, while allowingviruses and virus contaminants to flow through. In one embodiment, thepore size of the filter is between about 15 nm and about 50 nm.Filtration also can be carried out by nanofiltration using a 20 N or 35N filter (Planova, Asahi Kasei). In some embodiments, pre-filters areused to prevent fouling the nanofilter, e.g., an about 2 μM filter, or a0.2 p PVDF or PES membrane may be used

Polishing by Cation Exchange Chromatography

In some embodiments, the method further comprises, after chromatographywith hydroxyapatite (or tandem chromatography with hydroxyapatitefollowed by a mixed mode cation exchange/hydrophobic interaction resin),the optional step of polishing the sample comprising ADAMTS13 bychromatography on a cation exchange resin. In this step, theconductivity of the buffer comprising ADAMTS13 may be reduced prior topolishing, if necessary to achieve an appropriate conductivity for thecation exchange chromatography.

(a) Reducing Buffer Conductivity

After chromatography with hydroxyapatite or tandem chromatography withhydroxyapatite followed with a mixed mode cation exchange/hydrophobicinteraction resin, the buffer comprising ADAMTS13 protein may beprepared for cation exchange by reducing the conductivity of the buffer,e.g., by removing ionic components (e.g., sodium chloride). In someembodiments, buffer conductivity is reduced to less than about 5 mS/cmand/or the pH is reduced to about 6.0. The conductivity of the buffermay be reduced by any method known in the art, described herein, or ascan be appreciated by one of skill in the art, especially in light ofthe disclosures herein. Non-limiting examples includeultrafiltration/diafiltration (e.g., with crossflow cassettes orhollowfiber modules), gel filtration, dialysis, etc.

In one embodiment, the ADAMTS13 protein is prepared for cation exchangeby ultrafiltration/diafiltration with a membrane having an about 10 kDacut-off, against a cation exchange equilibration buffer (e.g., a buffercomprising 20 mM MES, pH 6.0 at room temperature). In some embodiments,the ultrafiltration/diafiltration membrane is a PES membrane, having anabout 10 kDa to an about 50 kDa cutoff, e.g., an about 20 kDa cutoff, anabout 30 kDa cutoff, an about 40 kDa cut-off, etc. Using such anapproach, the buffer pH may be reduced from about 8.0 to about 6.0;and/or the conductivity of the buffer may be reduced to below about 2mS/cm at room temperature. In some such embodiments, the buffer for thediafiltration may comprise 20 mM MES and may have a pH of 6.0 at roomtemperature and/or a conductivity of 0.6 mS/cm at room temperature. Insome embodiments, the conductivity of the buffer for diafiltration maybe identical, or substantially identical, to the cation exchangeequilibration buffer to be used.

In one embodiment, preparing the buffer comprising ADAMTS13 for cationexchange is performed by dialysis, e.g., using dialyzer hardwarecomprising hemodialysis modules, such as a hollowfiber hemodialysismodule (Aquamax series, PES chemistry of the Hollowfibers, EdwardsLifesciences, Unterschleiheim, Germany). Generally, about 2 m² of filterarea is used for about 5 L of sample; and the sample buffer and dialysisbuffer are run in reverse flow with respect to each other. In someembodiments, the dialysis consists of no more than two passes through asingle dialysis module. By “single dialysis module” is meant one unit orstructure through which dialysis is performed. A dialysis modulegenerally comprises an open-ended bundle of hollow fiber membrane pottedin a tubular housing to create two distinct flow chambers, lumen andextracapillary, each with inlet and outlet port access. A semi-permeablehollow fiber membrane separates the two chambers and selectively permitspassage based on size and concentration gradient of solutes whilerestricting other solutes from passing between the 2 chambers. Byoperating the module in a counter-current flow mode, the solutes passingthrough the membrane are quickly swept away and diluted into a largevolume of dialysate solution (“sweep”), maintaining the largestconcentration gradient possible. Accordingly, dialysis may be performedin a single pass sweep through a single dialysis module.

In some embodiments, a combination of these approaches is used, e.g.,UF/DF with dialysis may be used to effect concentration of the sampleand buffer exchange in preparation for polishing by cation exchangechromatography. In still other embodiments, buffer exchange may becarried out by anion exchange chromatography.

(b) Cation Exchange Chromatography

As indicated above, the method disclosed herein may optionally comprisepolishing the sample by chromatography on a cation exchange resin. Asused herein, the term “cation exchange resin” refers to any resinsuitable for cation exchange chromatography and that has a net negativecharge, e.g., due to a negatively-charged group (at neutral pH).Examples include, but are not limited to, a carboxyl group, acarboxymethyl (CM) group, a sulphoalkyl group (SP, SE), amethylsulfonate (S) group, a sulfated ester of cellulose, heparin, andthe like, and combinations thereof. This step generally is designed toconcentrate the ADAMTS13 product, put the product in a pre-formulationbuffer, and further reduce non-ADAMTS13 impurities, includingprocess-related impurities (e.g., host cell proteins, such as CHOproteins, host cell DNA, such as CHO DNA, reagents of thesolvent-detergent mixture, etc), as well as product-related impurities(e.g., aggregates and non-biologically active fragments of ADAMTS13).

In one embodiment, the cation exchange resin also has one or more of thefollowing features: large pores, perfusion flow behavior, and convectiveflow behavior. Non-limiting examples of commercially available cationexchange resins that may be used in the polishing step disclosed hereininclude POROS® S (Applied Biosystems), Convective Interaction Media(CIM®; BIA Separation), Toyopearl Gigacap S (Tosoh Bioscience,Montgomeryville, Pa.), Toyopearl Gigacap CM (Tosoh), Toyopearl SP(Tosoha), Toyopearl CM (Tosoh), MacroPrep S (Bio-rad, Hercules, Calif.),UNOsphereS (Bio-rad, Hercules, Calif.), MacroprepCM ((Bio-rad, Hercules,Calif.), Fractogel EMD SO3 (Merck), Fractogel EMD COO (Merck), FractogelEMD SE Hicap (Merck), Cellufine Sulfate (Chisso), CM and SP Trisacryl(Pall), CM and S HyperD (Pall), Mustang S (Pall), S and CM Sepharose CL(GE Healthcare), S and CM Sepharose FF (GE Healthcare), S and CM CAPTO™(GE Healthcare), MonoS (GE Healthcare), Source S (GE Healthcare), andthe like.

Chromatography on a cation exchange resin is a well-known method in theart. In some embodiments, the cation exchange column has a maximum loadof about 0.2 to about 0.5 mg ADAMTS13/mL. In a preferred embodiment, thecolumn is loaded with at least 0.3 mg ADAMTS13/mL. Generally, duringchromatography on a cation exchange resin, ADAMTS13 binds to the cationexchange resin and the buffer and certain impurities are allowed to flowthrough. The column to which ADAMTS13 is adsorbed then can be washed,e.g., to remove loosely-bound contaminants or impurities and/or toadjust the buffer in preparation for elution of ADAMTS13 from the cationexchange resin. ADAMTS13 then can be eluted in the eluate.

In some embodiments, the eluate obtained from the cation exchangechromatography step contains a higher amount of aggregates of ADAMTS13than desired. In some embodiments, for example, the eluate comprisesmore than about 15% aggregates, which are believed to be introducedafter the concentration and buffer exchange with the dialyzer step,and/or the cation exchange chromatography step.

To allow the production of ADAMTS13 with a significantly lowerpercentage of aggregates, certain conditions can be used with the cationexchange resin, as detailed further in FIG. 2 with respect to the cationexchange resin Poros S. For example, in some embodiments, a combinationcomprising purification by cation exchange chromatography followed byon-column solvent-detergent virus inactivation is used, for example asdescribed in more detail above. This combination preferably results inlower amounts of aggregates appearing with the ADAMTS13 polypeptide inthe eluate. In more preferred embodiments, the elution procedurecomprises a gradient elution (rather than a step elution), which canfurther remove aggregates of ADAMTS13, e.g., in the descending part ofthe elution peak. In even more preferred embodiments, the concentrationof Tween 80 in the elution buffer is greater than about 0.05%, forexample about 0.06%, about 0.07%, about 0.08%, about 0.09%, preferablyabout 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, or about0.15%. The increased concentration is believed to have a furtherstabilizing effect on ADAMTS13, further preventing formation of highmolecular weight structures during the elution of ADAMTS13 from theresin. By “stabilizing the ADAMTS13 protein” or “stabilizing effect onADAMTS13” is meant tending to promote the native structure of ADAMTS13,particularly, in an intact and/or monomeric form, or a substantiallyintact and/or monomeric form, rather than a fragmented or aggregatedform. Stabilizing may also refer to the tendency of the obtainedADAMTS13 to resist fragmentation, loss of native structure, and/oraggregation in the face of otherwise destabilizing conditions, such asvarying temperatures, varying pH, ionic strengths, and the like.

The ADAMTS13 protein also may be stabilized by a matrix used duringharvest, e.g., where virus inactivation by solvent-detergent treatmentis performed on a concentrated harvest. Furthermore, aggregates that doform can be removed by the later purification steps, such as capture onan anion exchange chromatography column (such as ANX Sepharose, asdescribed herein); and/or polishing by chromatography (such as tandemchromatography with Hydroxyapatite/Capto MMC, as described herein).

Using one or more modifications described above can result in an eluatecomprising lower amounts of aggregates of ADAMTS13 polypeptide. Forexample, in preferred embodiments, the eluate from the cationic exchangecolumn may comprise less than about 20%, less than about 18%, less thanabout 15%, less than about 12%, less than about 10%, or less than about5% aggregates.

A final step of chromatography on a cation exchange resin compriseseluting the ADAMTS13 protein with an elution buffer. In someembodiments, bound ADAMTS13 is eluted from the cation exchange resin byincreasing the ionic strength of the buffer. The buffer comprisingADAMTS13 used to chromatographically contact the cation exchange resingenerally has a conductivity of less than about 10 mS/cm, at roomtemperature, e.g., less than 5 mS/cm. Further, the buffer comprisingADAMTS13 used to chromatographically contact the cation exchange resingenerally has a pH of less than about 7.0, at room temperature e.g.,6.0. The elution buffer used to elute the ADAMTS13 protein from thecation exchange resin can have an ionic strength below such buffers. Theresin may also be washed with a buffer having a pH equal to, orsubstantially equal to, the pH of the intended storage buffer.

In a preferred embodiment, the ADAMTS13 protein is eluted from thecation exchange resin with a storage buffer. Generally, by storagebuffer is meant a buffer having a pH between about 5 and about 9, atroom temperature and comprising calcium, a buffering compound, and asalt. The pH of the storage buffer may be greater than about 7.0 (e.g.,about 7.5) at room temperature. The storage buffer may comprise lessthan about 10 mM Ca⁺⁺ (e.g., 2 mM Ca⁺⁺); the buffering compound may beselected from the group consisting of phosphate, tris, HEPES, histidine,imidazole, gly-gly, MES, tricine, acetate, and the like; and the saltmay be selected from the group consisting of NaCl, KCl, CaCl₂, MgCl₂,and the like. In a preferred embodiment, the storage buffer has a pH ofgreater than 7.0 and comprises less than 10 mM calcium ions, a bufferingcompound, and a salt. In a more preferred embodiment, the storage bufferfurther comprises a non-ionic detergent, e.g., about 0.01 to about 0.5%non-ionic detergent, e.g. 0.05% non-ionic detergent. In even morepreferred embodiments, the eluate is subjected to no subsequentconcentration nor buffer exchange steps following elution from thecation exchange resin with the storage buffer.

In one particular embodiment, Source S (GE healthcare) is used as thecation exchange resin of the polishing step, for example, a Source Scolumn with a bed height of about 20 cm. In such embodiments, the columnmay be activated with about 2 column volumes of about 2 M NaCl andequilibrated with about 6 column volumes of a buffer comprising about 20mM MES, about 10 mM NaCl, and about 2 mM CaCl₂, having a pH of about 6at room temperature. The buffer comprising ADAMTS13 may be contactedwith the column at a conductivity below about 5 mS/cm at roomtemperature, and the column subsequently washed with the equilibrationbuffer, and finally the eluate comprising ADAMTS13 protein collected.After collection, the eluate may be concentrated and buffer exchangedfor storage buffer, e.g., by anion exchange chromatography,diafiltration, ultrafiltration, dialysis, and the like.

In another particular embodiment, POROS® S is used as the cationexchange resin in polishing the sample comprising ADAMTS13 protein. Inthis embodiment, the buffer comprising ADAMTS13 used tochromatographically contact the cation exchange resin may have aconductivity of less than about 5 mS/cm and a pH between about 6.1 andabout 6.4. ADAMTS13 may be eluted using gradient elution, although stepelution may preferably provide a more concentrated product. If gradientelution is performed, two buffers may be used, e.g., a first buffer thathas a low salt content (e.g., little to no salt) and a second bufferthat has a higher salt content (e.g., about 500 mM) such that the eluatepool may have a salt concentration of about 200 mM. If step elution isperformed, the elution buffer may comprise a storage buffer, e.g., astorage buffer having about 300 mM NaCl, about 2 mM CaCl₂, about 20 mMhistidine, about 0.05% Tween 80, and may have a pH of about 7.5, at roomtemperature. In some such embodiments, no buffer exchange is necessaryafter the POROS® S step, i.e., the ADAMTS13 fractions obtained from thePOROS® S already are in a buffer and at a concentration suitable forstorage. In still other embodiments, the ADAMTS13 fractions obtainedfrom the POROS® S column may be subject to further concentrating and/orbuffer exchange steps.

Generally, purifying recombinant ADAMTS13 protein according to someembodiments of the methods disclosed herein yields compositions of pureADAMTS13 protein. In one embodiment, purifying recombinant proteinaccording to the disclosed method yields ADAMTS13 protein that is atleast about 90% pure, e.g., at least about 95% pure, e.g., at leastabout 98% pure, e.g., or at least about 99% pure. Yields of at leastabout 20% may be obtained according to some embodiments of the disclosedmethod. In one embodiment, the method provides yields of at least about5%, e.g., about 30%, e.g., about 10%, e.g., about 20%, e.g., about 40%,e.g., about 50%, e.g., about 60%, e.g., about 70%, e.g., about 80%,e.g., about 90%, or e.g., about 95%. In some embodiments, the methodprovides ADAMTS13 protein having specific activity ranging from about500 units/mg ADAMTS13 to about 1,000 units/mg ADAMTS13. In anotherembodiment, the method provides ADAMTS13 protein having a specificactivity ranging from about 1,200 units/mg ADAMTS13 UV 280 protein toabout 2,400 units/mg ADAMTS13 UV 280 protein. In another embodiment,wherein the recombinant ADAMTS13 protein is produced by CHO cellstransformed with recombinant ADAMTS13 nucleic acid, purifyingrecombinant ADAMTS13 according to the disclosed method produces acomposition that has less than about 1,000 ppm of host cell impurities.In some embodiments, the method provides at least about 2 mg/mL ADAMTS13protein in a storage buffer.

Compositions Comprising Recombinant ADAMTS13 Protein

The present invention further provides compositions comprisingrecombinant ADAMTS13 purified according to a method disclosed herein.The compositions disclosed herein may be useful for storage of purifiedrecombinant ADAMTS13. For example, in some embodiments, the purifiedADAMTS13 protein is stored frozen, e.g., at less than about −60° C. Thecompositions disclosed herein also may be useful for therapeuticadministration of the ADAMTS13 protein, and/or to prepare compositionsfor therapeutic administration, in particular, parenteraladministration. For example, in some embodiments, the purified ADAMTS13obtained according to methods described herein is in the form of a bulkdrug substance, i.e., in a form ready for formulation into compositionsfor therapeutic administration.

Accordingly, another aspect of the invention relates to pharmaceuticalcompositions where the purified recombinant ADAMTS13 protein is mixedwith excipient(s) or other pharmaceutically acceptable carriers. Inpreferred embodiments, the pharmaceutically acceptable carrier ispharmaceutically inert. A pharmaceutically inert carrier is one thatdoes not react, or does not react substantially, with the activepharmaceutical, and/or in particular, does not affect, or does notsubstantially affect, the desired pharmaceutical properties of theactive. The pharmaceutical compositions may be prepared in any mannerknown in the art e.g., by conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping,lyophilizing, and the like.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular administration, subcutaneous administration,intramedullary administration, intrathecal administration,intraventricular administration, intravenous administration,intraperitoneal administration, intranasal administration, and the like.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions comprising ADAMTS13 as the active ingredient in aneffective amount to achieve an intended purpose. An “effective amount”of ADAMTS13 as used herein can refer to that amount that augments,enhances, improves, increases, or produces a biological effect of nativeADAMTS13. Biological effects of native ADAMTS13 include vWF-cleavingprotease activity, based on the action of ADAMTS13 in cleaving vonWillebrand factor, a large protein involved in blood clotting. An“effective amount” will include an amount of ADAMTS13 that results indecreased levels of platelet aggregation, e.g., reducing levels in anindividual suffering from a blood clotting disorder to levels morecomparable to an individual not suffering from the blood clottingdisorder. Blood clotting disorders include, but are not limited to,thrombotic thrombocytopenic purpura (TTP) also known as Moschcowitzsyndrome, Upshaw-Schulman syndrome (familial form of TTP), and stroke.An “effective amount” also includes the amount to achieve a prophylacticand/or therapeutic benefit in treating one or more blood clottingdisorders and associated conditions. Determination of certain effectiveamounts is well within the capability of those skilled in the art.

The present invention provides methods, pharmaceutical compositions, andkits for treating and/or preventing blood clotting disorders andassociated conditions in animal subjects. The term “animal subject” asused herein includes humans as well as other mammals.

The term “treating and/or preventing” as used herein includes achievinga therapeutic benefit and/or a prophylactic benefit, respectively. Bytherapeutic benefit is meant the reversal or amelioration of theunderlying blood clotting disorder being treated. For example, in a TTPpatient, therapeutic benefit includes eradicating or ameliorating one ormore of the conditions and/or symptoms associated with TTP, such that animprovement is observed in the patient, notwithstanding the fact thatthe patient may still be afflicted with the underlying disorder. Forexample, treatment can provide a therapeutic benefit not only whenformation of thromboses is reduced or eradicated, but also when animprovement is observed in the patient with respect to symptoms thataccompany TTP, such as reduced headaches, lowered fever, and/or delayedkidney failure.

For prophylactic benefit, a pharmaceutical composition of the presentinvention may be administered to a patient at risk of developing a bloodclotting disorder, including, for example, a patient reporting one ormore of the symptoms or conditions commonly associated with bloodclotting disorders like TTP, even though a diagnosis may not yet havebeen made.

In addition to the active ingredient, pharmaceutical compositions maycomprise suitable pharmaceutically acceptable carriers such asexcipients and auxiliaries that facilitate processing of the activecompounds into preparations which can be used pharmaceutically.

Pharmaceutical formulations for parenteral administration generallycomprise aqueous solutions of the active ingredient in water-solubleform. In some embodiments, suspensions of the active may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase solubility of the active, e.g., toallow for the preparation of highly concentrated solutions.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

Pharmaceutical preparations for oral use can be obtained by combiningthe active with a solid excipient, optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gumsincluding arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid, or a salt thereof such as sodium alginate. Carriers may also beused that allow the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, solutions,suspensions, dragees, and the like, for oral and/or nasal ingestion by apatient to be treated.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active mixed with fillers or binders, such aslactose or starches; lubricants, such as talc or magnesium stearate;and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as oils, liquidparaffin, or liquid polyethylene glycol, with or without stabilizers.

Compositions comprising ADAMTS13 or other protein prepared according toa method described herein may formulated with a pharmaceuticallyacceptable carrier, placed in an appropriate container (or kit), andlabeled for treatment of an indicated condition.

EXAMPLES

The following examples are provided for illustrative purposes and arenot intended to limit the scope of the invention.

Example 1

FIG. 1 provides an exemplary method for purifying ADAMTS13 protein,according to certain embodiments of the invention as disclosed herein.In the Example provided, recombinant ADAMTS13 protein is purified fromsupernatant collected from culturing CHO cells comprising a recombinantADAMTS13 nucleotide sequence. In this example, the sample is cellculture supernatant comprising about 2 units/ml (approximately 2 μg/mL)ADAMTS13 protein.

As shown in FIG. 1, a sample comprising ADAMTS13 and non-ADAMTS13impurities may first be subject to an optional pre-enrichmentpreparation: (a) as shown in step 101, the sample may be concentrated byultrafiltration (about 10-fold to about 20-fold) and the bufferexchanged by diafiltration (molecular weight cut-off of about 30 kDa);and (b) as shown in step 102, the ADAMTS13 protein can be bond to andeluted from an anion exchange resin, prior to further enrichment.

Pre-Enrichment Preparation of the Sample

(a) Pre-Enrichment Ultrafiltration/Diafiltration (UF/DF)

As shown in FIG. 1, step 101, to optimize loading for pre-enrichmentanion exchange chromatography, the cell culture supernatant isconcentrated by about 10 fold to about 20 fold and diafiltrated using aPES membrane (about 30 kDa to about 50 kDa cutoff; Pall Omega) to alow-conductivity buffer containing calcium and zinc ions, which areconsidered to stabilize ADAMTS13. The buffer for the cell culturesupernatant diafiltration is 20 mM Tris, 0.1% Polysorbate 80, 85 mMNaCl, 2 mM CaCl₂, 5 μM ZnCl₂, with a pH of 7.7 at room temperature.

(b) Pre-Enrichment Anion Exchange Chromatography

As shown in FIG. 1, step 102, pre-enrichment anion exchangechromatography may be performed using ANX Sepharose Fast Flow low subfrom GE Healthcare. This anion exchange resin may be used according tothe following conditions (Tables 1-2).

Column load: max. 0.5 mg ADAMTTS13 Ag/ml resin; Bed height: 20 cm

TABLE 1 Column Flow rate Step Buffer Volume (CV) (cm/h) Column ANX-HS 2100 activation Equilibration ANX-Equi 6 100 Load Concentrated anddiafiltrated CCS Wash 1 ANX-W1 2.5 100 Wash 2 12.5% ANX-EluB/ 3 10087.5% ANXEluA Elution 12.5% ANX-EluB/ 11 100 gradient 87.5% ANXEluA to100% ANX-EluB Post elution ANX-HS 3 100

Alternatively, step elution can be used with 2.8 column volumes of abuffer comprising 48% ANX-EluB and 52% ANX-EluA, as detailed in Table 2.Other potentially suitable buffers are indicated also.

TABLE 2 Buffer Formulation ANX-Equi 20 mM Tris, 0.1% Polysorbate 80, 50mM NaCl, 2 mM CaCl₂, 5 μM ZnCl₂, pH = 7.7 (room temp.) ANX-W1 20 mMTris, 0.1% Polysorbate 80, 50 mM NaCl, pH = 7.7 (room temp.) ANX-EluA 20mM Na/K PO₄, pH = 7.0 (room temp.) ANX-EluB 20 mM Na/K PO4, 400 mM NaCl,pH = 7.0 (room temp.) ANX-HS 2 M NaCl

Different resins may be used from that indicated in FIG. 1, step 102.For example, POROS 50D and POROS 50PI from Applied Biosystems, FosterCity, Calif. can be used. The eluate from the pre-enrichment anionexchange chromatography using this resin can provide recombinantADAMTS13 with a purity of about 20% to about 70%, and the percent yieldafter this pre-enrichment preparation of the sample can be at leastabout 75%.

Enrichment of ADAMTS13

As shown in FIG. 1, steps 103 and 104, respectively, ADAMTS13 may thenbe enriched via polishing steps (a) and (b). The sample comprisingADAMTS13 is subjected to tandem chromatography, first withhydroxyapatite on a Hydroyapatite Type II column (Biorad, Hercules,Calif.), step 103, followed by mixed mode chromatography on a cationexchange/hydrophobic interaction resin CAPTO™ MMC (GE Healthcare), step104. More specifically, the eluate pool from the pre-enrichment anionexchange chromatography of step 102 is diluted 1:4 withhydroxyapatite-dilution buffer to reduce the conductivity to about 6mS/cm. The diluted eluate pool is subjected to tandem chromatographywith hydroxyapatite under conditions that allow a substantial portion ofADAMTS13 protein to flow through, step 103, followed by a mixed modecation exchange/hydrophobic interaction resin that binds ADAMTS13protein, step 104. The conditions for the tandem chromatography areprovided below and in Tables 3-4.

Column 1: Resin: Hydroxyapatite Type II (Biorad) (HA); load: max. 2 mgtotal protein/mL resin; bed height: 20-30 cm.

Column 2: Resin: Capto MMC (GE Healthcare); load: 3-6 mg ADAMTS13/mlresin; bed height: 10 cm.

Ratio Column volume HA:MMC=10:1.

TABLE 3 Column Flow rate Step Buffer Volume (CV) (cm/h) ActivationMMC-Elution 3 (MMC) 50 (MMC) (MMC) Equilibration HA-Equi. 4 (HA) 50 (HA)Equilibration HA-Equi. 1 (HA) 50 (HA) Load Diluted Capture Eluate 20-30L 30 (HA) (1:5 diluted with HA- Equi) <6 mS/cm conductivity Re- HA-Equi.0.5 (HA) 30 (HA) equilibration Wash 1 MMC-Equi. 3 (MMC) 50 (MMC) (MMC)Wash 2 MMC-Wash 4 (MMC) 50 (MMC) (MMC) Elution 75% MMC elution buffer/ 4(MMC) 50 (MMC) (MMC) 25% MMC wash buffer

TABLE 4 Buffer Formulation Conductivity HA-Dilution 20 mM Na/K PO₄, pH7.0 (room temp.) TANDEM-Equi. 20 mM Na/K PO₄, 25 mM About 5.5 mS/cmNaCl, pH 7.0 (room temp.) (room temp.) MMC-Wash 50 mM Na/K PO₄, 160 mMAbout 16.5 mS/cm NaCl, pH 8.0 (room temp.) (room temp.) MMC-Elution 50mM Na/K PO₄, 1000 About 93 mS/cm mM NaCl, pH 8.0 (room (room temp.)temp.) HA-Elution 300 mM K PO₄, pH 7.0 About 33 mS/cm (room temp.) (roomtemp.)

The percent yield ADAMTS13 after enrichment by tandem chromatography maybe at least 60%.

Virus Inactivation

As shown in FIG. 1, step 105, the sample can be subjected tosolvent-detergent treatment to inactivate contaminating viruses or viralparticles; and/or the sample is filtered to remove such viruses or viralparticles. Also as shown in FIG. 1, virus inactivation step 105 can becarried out at various points in the procedure, for example, before thetandem chromatography steps 103 and 104, or after a step involvingconcentration and buffer exchange, step 106, described below.

For virus inactivation by solvent-detergent treatment, the sample istreated with a solvent-detergent mixture comprising 1% TRITON X-100®,0.3% Tri-N-butylphosphate, and 0.3% polysorbate 80, for 30 minutes atabout 12° C. to about 16° C. (specifically to inactivate lipid-envelopedviruses). Additional details are provided below in Example 2.

Alternatively, or in addition, the sample is subjected to filtration,e.g., nanofiltration through a 0.2 μm particle filter. For example, themixture after solvent-detergent treatment is diluted with 1 volume of apolishing equilibration buffer, described below, and filtered through a0.2 μm PVDF or PES membrane. Filtration can be carried out before and/orafter solvent-detergent treatment. Filtration after the treatment may beused to remove particulate matter that may have formed during thetreatment. Filtration also can be carried out by nanofiltration using a20 N filter (Planova, Asahi Kasei), as also shown in FIG. 1, where thevirus inactivation step 105 is carried out before the tandemchromatography steps 103 and 105. A further virus inactivation step 105can be carried out after the sample has been polished by cation exchangechromatography, as described below.

The percent yield ADAMTS13 from this virus inactivation may be at least95%.

Polishing by Cation Exchange Chromatography

Following enrichment, the ADAMTS13 may be polished by chromatography ona cation exchange resin, and the conductivity of the buffer comprisingADAMTS13 may be reduced prior to polishing, to achieve an appropriateconductivity for the cation exchange chromatography. Accordingly,post-enrichment steps may involve (a) reducing buffer conductivity;followed by (b) cation exchange chromatography.

(a) Reducing Buffer Conductivity

As shown in FIG. 1, step 106, preparation for cation exchangechromatography may involve concentration and buffer exchange, usingUF/DF, with a cut-off of 10 kDa, and Dialyzer hardware. In theillustrated embodiment, the Dialyzer hardware used for buffer exchangeinvolves a hollowfiber hemodialysis module (Aquamax series, PESchemistry of the Hollowfibers, Edwards Lifesciences, Unterschleiheim,Germany) having 0.3-1.9 m² filter area. During operation, the followingparameters are monitored on-line: pressure (before the module, after themodule, and trans-membrane pressure), conductivity, and temperature. Thedialyzer cartridge is connected with two pumps, one feeding the sample(through the hollow fibers) and one feeding the dialysis buffer(surrounding the hollow fibers, in reverse flow direction).Approximately 2 m² of filter area is used for about 5 L of sample; andthe fluid flow is fixed in the following way: 40 ml/min (sample flow or20 ml/min/m² filter area), 60 ml/min (dialysis buffer flow, reverseflow). Before and after dialysis, the hollowfiber module is rinsed withdialysis buffer and the post-dialysis rinse added to the collectedproduct. After dialysis, the sample has about the same volume as before,although it is slightly concentrated.

The percent yield ADAMTS13 after reducing the buffer conductivity inthis way may be about 90%.

In other embodiments, buffer exchange may be carried out by anionexchange chromatography on ANX Sepharose-FF low sub, as in step 102.

Cation Exchange Chromatography

As shown in FIG. 1, step 107, after enrichment of ADAMTS13 protein (andthe optional concentration and buffer exchange step 106 and/or the virusinactivation step 105), the sample may be polished by cation exchangechromatography. The buffer comprising ADAMTS13 protein is polishedeither on a Source S column (GE Healthcare) or a POROS® S column, suchas on POROS® 50 HS column (Applied Biosystems).

The conditions for polishing on the Source 30S column are provided inTable 5 and buffers for the polishing step are provided in Table 6.

Resin: Source 30 S (GE Healthcare); Column load: max. 0.2 (0.5) mgADAMTS13/ml resin; Bed height: 20 cm.

TABLE 5 Column Flow rate Step Buffer Volume (CV) (cm · h) Column 2 MNaCl 2 32 activation Equilibration SOS-Equi. 6 32 Load 32 Wash SOS-Equi.3 32 Elution 100% SOS-Equi/ 5 19 (gradient) 0% SOS-Elu. to 0% SOS-Equi./100% SOS-Elu. Post elution SOS-Elu. 3 32

TABLE 6 Buffer Formulation Comments SOS-Equi. 20 mM MES, pH 6.0 Buffermay contain 10 mM (room temp.) NaCl, 2 mM CaCl₂ SOS-Elu. 20 mM MES, 500mM NaCl, 2 mM CaCl₂, pH 6.0 (room temp.)

The eluate pool from the Source S column is concentrated anddiafiltrated against storage buffer.

The conditions for polishing on the POROS® S column are provided inTable 7 and buffers for the polishing step on are provided in Table 8.

Resin: POROS® S (Applied Biosystems, Foster City, Calif.); Column load:max. 12 mg ADAMTS13/ml resin; Bed height: 20 cm.

TABLE 7 Column Flowrate Step Buffer Volume (CV) (cm/h) Column activation2M NaCl 5 CV 50 Equilibration Poros 10 CV (until pH and 50 Equi.conductivity give flat and stable signals) Load MMC-Eluate conductivityless that 5 32 after solvent- mS/cm (room temp.) detergent treatment anddilution Re-equilibration Poros Equi. 5 CV 32 Wash 1 Poros Wash 1 5 CV32 Wash 2 Poros Wash 2 7 CV 32 Elution Poros Elu. 5 CV 19 Post elution2M NaCl 3 CV 32

TABLE 8 Poros Equi. 20 mM MES Acid, 30 mM NaCl, 0.1% Tween 80, pH 6.0(room temp.), about 3.9 mS/cm conductivity at 25° C. Poros 20 mML-Histidine, 5 mM NaCl, 2 mM CaCl₂, 0.05% Wash 1 Tween 80, pH 6.0 (roomtemp.), about 1.9 mS/cm conductivity at 25° C. Poros 20 mM L-Histidine,5 mM NaCl, 2 mM CaCl₂, 0.05% Wash 2 Tween 80, pH 7.5 (room temp.), 1.9mS/cm conductivity at 25° C. Poros Elu. 20 mM L-Histidine, 300 mM NaCl,2 mM CaCl₂, 0.05% Tween 80, pH 7.5 (room temp.), about 18 mS/cmconductivity at 25° C.

The eluate pool from the POROS® S column is concentrated anddiafiltrated against storage buffer.

The percent yield ADAMTS13 after this further polishing step may be atleast about 70%, and after the buffer exchange, at least about 90%.

As shown in FIG. 1, step 108, a purified ADAMTS13 protein is obtained inaccording to the method described above. The ADAMTS frozen and stored,e.g., at less than about −60° C. The yield of the entire process may beabout 22% to about 24% or more.

Example 2

FIG. 2 provides a summary of various conditions that can be used withcation exchange chromatography step 107 of FIG. 1. In particular,comparison of the ADAMTS13 product obtained from the various runsindicates that the conditions of FIG. 2C reduce contaminatingaggregates.

As shown in FIG. 2A, variation A is a combination of viral inactivationusing solvent-detergent (S/D) treatment, as discussed in more detailbelow, followed by a cation exchange chromatography on Poros S applyinga step elution. As shown in FIG. 2B, variation B involves cationexchange chromatography on Poros 50S, with step dilution, but withoutbeing preceded by virus inactivation. Both variations A and B can beperformed according a procedure outlined in Table 9.

TABLE 9 Buffer Flow volume rate (CV) Buffer Composition (cm/h)Observations Activation 5 2 M NaCl 50 Equilibration 6 20 mM MES Acid, 30mM 50 NaCl, pH 6.0 (room temp.) Product about 12 S/D treated and diluted32 Column load max. 6 loading product solution mg ADAMTS13/ml resin Wash1 10 20 mM MES acid, 30 mM 32 NaCl, pH 6.0 (room temp.) Wash 2 8 20 mMHistidine, 30 mM 32 NaCl, 2 mM CaCl₂, 0.05% Tween 80, pH 7.0 (roomtemp.) Step elution 5 20 mM Histidine, 200 mM 25 Pooling starts afterNaCl, 2 mM CaCl₂, 0.05% the UV₂₈₀ signal rises Tween 80, pH 7.5 (roomtemp.) significantly and pooling ends after the UV₂₈₀ signal drops below5% of the UV₂₈₀ signal at the peak maximum (approximately 1 CV)

In variation A, the conditioned (dialyzed) eluate from step 106 issubjected to a solvent-detergent virus inactivation step 105. The eluateis first filtered through a filter with 0.2μ pore size to removeparticular matter. Then the filtrate is supplemented with asolvent-detergent mixture to final concentrations of 1% Triton X-100,0.3% tri-n-butyl phosphate and 0.3% Polysorbate 80 (Tween 80) from stocksolutions. The inactivation is performed at temperatures ranging fromabout 12° C. to about 25° C. in a time frame of about 30 minutes toabout one hour under slight stirring or shaking. The inactivation isstopped by diluting the solution with one volume of cold dilution buffer(20 mM MES, pH 6.0, room temp.). To protect the column, thesolvent-detergent treated and diluted solution is filtered again with a0.2μ filter, for example, to remove particulate matter that may haveformed during the virus inactivation treatment.

The solvent-detergent inactivated and diluted product solution is thensubjected to cation exchange chromatography step 107 on Poros 50HS,using step dilution. Chromatographic details are outlined in Table 9above. The resulting eluate pool provides the ADAMTS13 protein in theform of bulk drug substance, which can be stored frozen at less than−60° C.

In variation B, the cation exchange chromatography step 107 is carriedout on the conditioned (dialyzed) eluate from step 106, without thesolvent-detergent virus inactivation step 105. Details for the cationexchange chromatography are as detailed above.

As shown in FIG. 2C, variation C is a combination involving purificationby cation exchange chromatography on Poros 50HS, using gradient elution,followed by on-column solvent-detergent virus inactivation. Thisvariation surprisingly reduces aggregates otherwise found with purifiedADAMTS13 protein. Chromatographic details that may be used withvariation C are outlined in Table 10 below.

TABLE 10 Buffer Flow volume rate (CV) Buffer composition (cm/h) CommentsActivation 5 2 M NaCl 50 Equilibration 6 20 mM MES acid, 30 mM 50 NaCl,pH 6.0 (room temp.) Product about 6 Dialyzed eluate pool of the 32Column load max. 6 loading Capto MMC purification mg ADAMTS13/ml resinWash 1 10 20 mM MES acid, 30 mM 32 NaCl, pH 6.0 (room temp.) Wash 2 1.520 mM MES acid, 30 mM 32 NaCl, 1% Triton X-100, 0.3% TNBP, 03% 0.3%Tween 80, pH 6.0 (room tmep.) Wash 3 2.1 20 mM MES acid, 30 mM 20 S/DTreatment: 1 NaCl, 1% Triton X-100, hour contact time 0.3% TNBP, 03%0.3% with S/D chemicals Tween 80, pH 6.0 (room temp.) Wash 4 10 20 mMMES acid, 30 mM 32 Removal of S/D NaCl, pH 6.0 (room temp.) chemicalsWash 5 8 20 mM Histidine, 30 mM 32 Conditioning column (buffer A) NaCl,2 mM CaCl₂, 0.1% for elution Tween 80, pH 7.0 (room temp.) Step Elution10 Gradient from 100% buffer 32 Pooling starts after A to 100% buffer B(20 mM the UV₂₈₀ signal rises Histidine, 300 mM NaCl, 2 significantlyand mM CaCl₂, 0.1% Tween 80, pooling end after the pH 7.5 (room temp.)within UV₂₈₀ signal drops 10 CV below 5% of the UV₂₈₀ signal at the peakmaximum (about 2-3 CV)

In variation C, the load material is the eluate pool of the cationexchange polishing step 104 and preferably has a conductivity below 4.5mS/cm, achieved by dialysis or buffer exchange by gel filtration.Notably, the cation exchange chromatography on Poros S is adapted toinclude an on-column solvent-detergent treatment, which involves virusinactivation of virus immobilized on the chromatographic column, asdiscussed above. The on-column virus inactivation comprises a wash forone hour with the solvent-detergent mixture at 2° C. to 10° C. After theon-column treatment, the wash buffer is changed to efficiently wash outsolvent-detergent chemicals prior to elution.

Elution is changed from step elution with 200 mM NaCl to a gradientelution, which is believed to facilitate separation of monomeric andoligomeric species of ADAMTS13, particularly in the descending part ofthe elution peak. Aggregates are removed in the late eluting fraction,thereby further removing aggregates otherwise found with purifiedADAMTS13 protein. As a further adaptation to stabilize monomericADAMTS13 protein, the concentration of Tween 80 in the elution buffer isincreased from 0.05% to 0.1% in the wash and elution buffers. This isbelieved to further prevent formation of aggregates during the elutionof ADAMTS13 from the Poros S resin. The details of the chromatographicprocedure on Poros S, including virus inactivation by on-columnsolvent-detergent treatment, are outlined in Table 10 above.

As shown in FIG. 2D, variation D serves as a control. In variation D,step 107 polishing via cation exchange chromatography is performed onPoros 50 HS again with gradient elution and increased Tween 80 in theelution buffer, but without on-column virus inactivation bysolvent-detergent treatment. A virus inactivation solvent-detergenttreatment step 105 is performed instead on concentrated harvest prior tocation exchange. The chromatographic details are provided in Table 11below.

TABLE 11 Buffer Flow volume rate (CV) Buffer composition (cm/h) CommentsActivation 5 2 M NaCl 50 Equilibration 6 20 mM MES acid, 30 mM 50 NaCl,pH 6.0 (room temp.) Product about 6 Dialyze eluate pool of the 32 Columnload max. 6 loading Capto MMC purification mg ADAMTS13/ml resin Wash 110 20 mM MES acid, 30 mM 32 NaCl, pH 6.0 (room temp.) Wash 2 10 20 mMHistidine, 30 mM 32 Conditioning column (buffer A) NaCl, 2 mM CaCl₂,0.1% for elution Tween 80, pH 7.0 (room temp.) Step elution 10 Gradientfrom 100% buffer 32 Pooling starts after A to 100% buffer B (20 mM theUV₂₈₀ signal rises Histidine, 300 mM NaCl, 2 significantly and mM CaCl₂,0.1% Tween 80, pooling ends after the pH 7.5 (room temp.) within UV₂₈₀signal drops 10 CV below 5% of the UV₂₈₀ signal at the peak maximum(about 2-3 CV)

In variation D, the load material is the eluate pool of the cationexchange polishing step 104 and preferably has a conductivity of below4.5 mS/cm, achieved by dialysis or buffer exchange by gel filtration.The cation exchange chromatography step 107 on Poros S again usesgradient elution, instead of step elution, as well as 0.1% Tween 80 inthe wash and elution buffers, as described above. The details of thechromatographic procedure on Poros S with gradient elution are outlinedin Table 11, above.

Example 3

Experiments at lab scale, using 100 L fermenter scale, were performedwith the on-column solvent-detergent virus inactivation, to determinepotential impact of this procedure on the performance of cation exchangechromatography step 107. The data are presented in Table 12.

TABLE 12 CHO HCP Yield Poros S* Specific impurity Solvent- % A13activity ng CHO ng CHO Aggregates detergent % A13 Frets Units/mgHCP/Unit HCP/mg % % % Sample procedure Ag Units A13 Ag A13 A13 Agmultimers dimers monomer 1 Solvent- 99 100 696 0.49 346 9.7 7.0 83.3detergent treatment immediately before chromatography on Poros S(variation A) 2 no solvent/ 133 154 894 0.58 519 1.3 4.6 94.1 detergenttreatment (variation B) 3 Solvent- 89 95 931 0.60 561 1.0 2.3 96.7 4detergent 87 117 905 0.39 354 1.0 3.7 95.3 treatment on-column (Poros S)(variation C) 5 Solvent- 65 93 764 0.21 163 0.8 1.5 97.7 6 detergent 81108 845 0.38 324 0.7 1.5 97.8 7 treatment at 66 131 741 0.31 231 0.2 1.198.7 the concentrated harvest (variation D) *Yields above 100% reflectsan assay problem with the chromatographic load fraction on Poros 50S.A13 Ag: ADAMTS13 antigen A13 Frets: ADAMTS13 Frets Units CHO HCP:Chinese hamster ovary host cell proteins

As shown in Table 12, performing virus inactivation viasolvent-detergent treatment in solution, prior to cation exchangechromatography on Poros S, can result in the formation of high amountsof aggregates (Variation A of FIG. 2A and Sample 1). If thesolvent-detergent treatment is omitted and the same procedure carriedout, aggregate formation is significantly reduced (Variation B of FIG.2B and sample 2).

Performing the solvent-detergent treatment on-column, that is,contacting the ADAMTS13 with the solvent-detergent mixture while it isimmobilized on the surface of the resin, also can prevent the formationof aggregates. In addition, the small amounts of aggregates that do formcan further be removed by gradient elution in the late eluting fraction(Variation C of FIG. 2C; samples 3 and 4).

For comparison, a standard solvent-detergent treatment in solution iscarried out the purification process, followed by cation exchangechromatography on Poros S without solvent-detergent treatment neitherimmediately before nor on-column (Variation D of FIG. 2D, samples 5, 6,and 7). This procedure also can produce ADAMTS13 with a low content ofaggregates.

All patents and patent publications referred to herein are herebyincorporated by reference.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

The invention claimed is:
 1. A method for purifying recombinant adisintegrin-like and metallopeptidase with thrombospondin type I motif13 (ADAMTS13) protein from a sample comprising ADAMTS13 protein andnon-ADAMTS13 impurities, the method comprising the steps of: (a)chromatographically contacting the sample with hydroxyapatite underconditions that allow said ADAMTS13 protein to appear in a flow throughfraction from said hydroxyapatite by said ADAMTS13 protein not bindingto said hydroxyapatite while said non-ADAMTS13 impurities are retainedon said hydroxyapatite; (b) subsequently chromatographically contactingsaid flow through fraction with a mixed mode cation exchange/hydrophobicinteraction resin that hinds said ADAMTS13 protein; and (c) eluting saidADAMTS13 protein from said mixed mode cation exchange/hydrophobicinteraction resin with an elution buffer.
 2. The method according toclaim 1, further comprising chromatographically contacting said samplewith an anion exchange resin and eluting said ADAMTS13 protein from saidanion exchange resin before step (a) chromatographic contact with saidhydroxyapatite.
 3. The method according to claim 1, further comprisingconcentrating said ADAMTS13 protein in said sample by ultrafiltration;and stabilizing said ADAMTS13 protein by diafiltration exchange into abuffer comprising calcium ions and zinc ions before step (a)chromatographic contact with said hydroxyapatite.
 4. The methodaccording to claim 1, further comprising, following contact with in step(a) said hydroxyapatite or in step (g) said mixed mode cationexchange/hydrophobic interaction resin, a preparing step (d) forpreparing said ADAMTS13 protein for cation exchange by reducing bufferconductivity.
 5. The method according to claim 4, wherein said preparingstep (d) is performed by ultrafiltration/diafiltration.
 6. The methodaccording to claim 4, wherein said preparing step (d) is performed bydialysis, said dialysis being performed once or twice.
 7. The methodaccording to claim 5, wherein said preparing step (d) is performed bygel filtration.
 8. The method according to claim 4, further comprisingsubjecting said ADAMTS13 protein to at least one virus inactivation orvirus removal step (e), wherein said virus inactivation or virus removalstep is performed after said preparing step (d).
 9. The method accordingto claim 1 or 4, further comprising subjecting said ADAMTS13 protein toat least one virus inactivation or virus removal step (e).
 10. Themethod according to claim 9, Wherein said virus inactivation step (e)comprises adding a solvent-detergent mixture comprising a non-ionicdetergent and an organic solvent to said ADAMTS13 protein.
 11. Themethod according to claim 9, wherein said virus removal step (e)comprises filtering said ADAMTS13 protein with a nanofilter to removeviruses and/or viral particles.
 12. The method according to claim 10,wherein said ADAMTS13 protein is immobilized during said virusinactivation step (e).
 13. The method according to claim 10, whereinsaid solvent-detergent mixture comprises 1% TRITONX-100, 0.3%Tri-N-butyl phosphate, and 0.3% TWEEN
 80. 14. The method according toclaim 12, wherein said ADAMTS13 protein is in step (e) immobilized on acation exchange resin.
 15. The method according to claim 14, furthercomprising eluting said ADAMTS13 protein from said cationic exchangeresin using gradient elution, said gradient elution comprising using afirst buffer having low salt content and a second buffer having highersalt content.
 16. The method according to claim 14, further comprisingeluting said ADAMTS13 protein from said cationic exchange resin usingstep elution.
 17. The method according to claim 16, wherein said stepelution comprises eluting said ADAMTS13 protein from said cationicexchange resin with a storage buffer.
 18. The method according to claim17, wherein said storage buffer has a pH of greater than 7.0 andcomprises less than 10 mM calcium ions, a buffering compound, 0.05%non-ionic detergent, and a salt.
 19. The method according to claim 17,wherein there is no ultrafiltration, diafiltration or buffer exchangestep subsequent to the eluting step from the cation exchange resin withthe storage buffer.